CN1526025A - Genes expressed in breast cancer as prognostic and therapeutic targets - Google Patents

Abstract

Methods are disclosed for, determining the endocrine responsiveness of breast carcinoma and treating and monitoring the progression of breast carcinoma based on genes which are differentially expressed in breast tumors. Also disclosed are methods for identifying agents useful in the treatment of breast carcinoma, methods for monitoring the efficacy of a treatment for breast carcinoma, methods for inhibiting the proliferation of a breast carcinoma, and breast-specific vectors including the promoters of the disclosed genes.

Description

The gene of expressing in the mammary cancer as prognosis and treatment target

The application requires the right of priority of the U.S. Provisional Application submitted to May 16 calendar year 2001 number 60/291,428, and to incorporate this provisional application into be reference to integral body here.

Invention field

The present invention relates to the monitoring of cancer, prognosis and methods of treatment.Particularly, the present invention relates to the purposes of gene expression analysis, be used to detect mammary cancer the responsiveness of endocrine therapy and the effectiveness that helps to select the mammary cancer therapy or monitor various mammary cancer therapies.

Background technology

Mammary cancer is the modal cancer that influences American Women.Only, just diagnose the new mammary cancer case of about 200,000 examples every year, and about 44,000 women will die from this disease in the U.S..During women's all one's life, mammary cancer will take place with the probability of 12.5% (having 1 among per 8 women), accounts for 32% of woman cancer case.It is second major cause of women's cancer mortality after lung cancer.Male breast carcinoma accounts for the about 1% of all new cases, with women with breast cancer similar natural history is arranged.Although the sickness rate of mammary cancer reduces lentamente now, mortality ratio in the past few decades but keeps constant.The whole world, the annual about 100 ten thousand new mammary cancer cases of diagnosing.Usually, there is the highest sickness rate in rich western countries, and there is minimum sickness rate in developing country.

Although the cause of disease of mammary cancer still is unknown, a large amount of Hazard Factor have been identified.For example along with the increase at age, the sickness rate of mammary cancer increases significantly; In the U.S., the women who suffers from breast cancer more than 50% is more than 60 years old.Other Hazard Factor are less age when menarche and the big age when climacteric.

Recently, find the tumor suppressor gene of supposition, the sudden change among BRCA-1 and the BRCA-2 may be the cause of disease of most of mammary cancer.Women with these sudden changes usually has positive family history, and in all patient with breast cancers, have the 5% clear pattern of having observed autosomal dominant inheritance (referring to, Cecil, " Textbook of Medicine ", Goldman and Bennett, editor, Saunders Co., Philadelphia, PA).

Cancer by stages when the treatment of mammary cancer and net result depended on oncological pathology and treatment.The most frequently used system by stages is the TNM system.This system according to the existence of tumour size, lymph node involvement degree and transfer determine the state of cancer and stage (referring to, american cancer joint committee: AJCCCancer Staging Handbook, Lippincott-Raven, Philadelphia, PA (1998)).The percentage measurement result that carcinoma stage during detection determined do not recur in 10 years.This is after removing original tumour by mastectomy or lumpectomy, does not experience the patient's of original cancer return percentage ratio in 10 years.

The symptom variation of mammary cancer is very big, and depends on the position of primary tumo(u)r and existence, position and the scope of size and transfer.Yet can comprise one or more following symptoms: the palpable mammary gland tumor in one or both sides, nipple discharge, skin of breast change, mastalgia.This may have also may not have the natural cycle (promptly being accompanied by the menstrual period), the nipple discharge of blood sample or water sample, palpable oxter lump or other lymph node involvement sign.

If primary tumo(u)r shifts, can in any tract of health symptom appear so.Modal transfer site is a regional area, and promptly the wall of the chest and/or regional nodes (20-40%), bone (60%), lung (being pernicious transudate and/or material injury) are (15-25) and liver (10-20%).Central nervous system (CNS), spinal cord or other bone shift and the pia-arachnoid transfer can cause part or diffustivity pain, particularly have a back ache and neurological symptom or dysfunction comprise numbness (parathesias), paraplegia, sensation reduction or forfeiture and hypercalcemia.Following CNS to involve modal is epileptic seizures, headache, the change of the mental status or or even paralysis or apoplexy.Hepatic metastases can cause liver failure, with the liver functional testing, jaundice and/or other hepatic insufficiency sign that raise.Involving of lung can cause expiratory dyspnea, pneumonia or other respiratory symptom.Because tumour cell can be attacked in any tissue of health and increase, so above-mentioned symptom all is common having or not having in the mammary cancer of transfer, but similar all syndromes may appear in the patient who suffers from breast cancer also.

In the patient with breast cancer, identified a large amount of prognosis factors, comprise degree, the axillary lymph footing amount of involving and the size of tumour of tumor by local invasion and attack, and these factors have been included in the above-mentioned system by stages.

Yet an important predictive factor is the expression of estrogen receptor alpha (ESR1) on tumor cell surface in the mammary cancer.Estrogen receptor (ER) is the transcription factor that part orders about, the expression of the several genes (comprising somatomedin, hormone and oncogene) that its adjusting is important to growth of breast cancers (referring to, Gronemeyer, Ann.Rev.Genetics, 25 volumes, 89-123 page or leaf (1991); Dickson ﹠amp; Lippman, " The Molecular Basis of Cancer ", Mendelsohn edits; Howley, Israel ﹠amp; Liotta edits, 358-384 page or leaf, W.B.Saunders Co., Philadelphia, PA (1994)).The morbidity that is expressed in mammary cancer of ER and keep in play an important role.About 2/3rds tumour is the ESR1 positive (referring to, Lippman etc., Cancer, the 46th volume, 2838-2841 page or leaf (1980)) among the patient with breast cancer.These ER positive tumors of about 50% are estrogen-dependents, and endocrine therapy produced reply (referring to, Manni etc., Cancer, the 46th volume, 2838-2841 page or leaf, (1980); Jensen, Cancer, 47 volumes, 2319-2326 page or leaf, (1981)).The mammary cancer that occurs in the postmenopausal women usually is that (referring to, Iglehart, " Textbook of Surgery " the 14th edition, Sabiston edits the ER male, 510-550 page or leaf, W.B.Saunders, Philadelphia, PA (1991).The significantly more ER of the normal breast epithelium of many tumour expression ratios in these tumours (referring to, Ricketts etc., Cancer Res., 51 volumes, 1817-1822 page or leaf (1991)).

The ESR1 gene is across 140Kb, and 8 exons that produced 6.3Kb RNA by montage are formed, this RNA coding molecule amount be 66 kilodaltons have 595 amino acid whose protein (referring to, Walter etc., Proc.Natl.Acad.Sci.USA, the 82nd volume, 7889-7893 page or leaf; With Ponglikitmongkoli etc., EMBO J., 7 volumes, 3385-3388 page or leaf).

The patient who does not express ER with primary lesion compares, and the survival rate that primary lesion is expressed the patient of ESR1 improves 5-10% at least.

In addition, very importantly, the existence of ESR1 tends to indicate the positive response to the assisting therapy of internal secretion form of therapy in the primary lesion.The purpose of endocrine therapy is the activation that stops ER on the tumour cell, the growth of tumour cell piece and hyperplasia are reduced or stops.

Utilized several different methods to stop the activation of ER among the patient with breast cancer.The most widely used medicament is estrogen antagonist agent such as Tamoxifen (tamoxifen), and it suppresses estrogen effect in the malignant cell level.Although Tamoxifen has the effect of agonist and antagonist to ER, it works as the estrogen antagonist medicine.Traditionally, this medicine is advanced breast cancer patient's a primary methods of treatment.

Yet, unfortunately, for the patient of the positive mammary cancer of ER in late period, only be about 50% (referring to, Clark etc., Semin.Oncol., 15 volumes, augment 1,20-25 page or leaf, (1988) at 2 phases) to the response rate of Tamoxifen.Under many situations of replying that do not exist Tamoxifen, seem that growth of tumor becomes not to be subjected to estrogenic control, and the application of estrogen antagonist medicine is with inoperative.Yet, unexpectedly, about 1/3rd Tamoxifen resistance patients will produce the reduction of endogenous estrogen level and reply (referring to, Dombernowsky etc., J.Clin.Oncol., 16920 volumes, 453-461 page or leaf, (1998); With Crump etc., Breast Cancer Res.Treat., 44 volumes, 3 phases, 201-210 page or leaf, (1997)).In postclimacteric patient, with selectivity on-steroidal aromatase inhibitor letrozole (letrozole) (Femara ^TM) can reach this purpose (referring to Dombernowsky etc., above).Femara is a kind of aromatase inhibitor, and it is by in conjunction with aromatase enzyme and suppress it and adrenal androgen is converted into oestrogenic hormon works.

In addition, also utilized by reducing other medicament that the estrogen concentrations that can arrive target cell plays clinical effect.These medicaments comprise progesterone, as megestrol and Veramix, LHRH, male sex hormone and other aromatase inhibitor, as anastrozole (Anastrozole) (referring to, Litherland etc., Cancer Treatment Reviews, 15 volumes, 183-194 page or leaf (1988)).

Therefore, usually, tumour is that ER male patient is the good candidate of endocrine therapy.Yet, discuss as above-mentioned, only 30-70%ESR1 male malignant tumour will be to endocrine therapy, for example the treatment of estrogen antagonist or estrogen deprivation produce and reply (referring to, Clark etc., Semin.Oncol., 15 volumes, 20-25 page or leaf (1988); With Lutherland etc., Cancer Treatment Reviews, 15 volumes, 183-194 page or leaf, (1988)).The molecular basis of the positive malignant tumour of the ESR1 of anti-endocrine therapy is also not quite understood.

By measuring the gene PgR (PGR) and the trifolium factor 1 (TFF1) of estrogen regulating, the expression of (being also referred to as PS2) has carried out increasing the trial of biomarker to the indication ability of endocrinotherapy for breast cancer.The existence that has deixis and activation ER of one of these protein, and these two kinds of protein predictive biomarker that all is endocrinotherapy for breast cancer.Be worth although the application that PGR expresses has improved the predictive of independent ESR1, under transfer case, 20% expression ER and the tumour of PGR still can not produce endocrine therapy and reply.Similarly, although TFF1 is relevant with good prognosis, and indication is to the positive response of hormonotherapy, but also do not prove its be enough to as the conventional assessment of mammary cancer the predictive biomarker (referring to, Ribieras etc., Biochem.Biophys.Acta., F-61-F77 volume, 1378 pages, (1998)).

Although the method for the state of the existence of ER and PGR and TFF1 in the assessment breast cancer tumour cell, predict that as part binding assay or being applied in of immunohistochemistry (IHC) endocrine therapy is valuable in replying based on kytoplasm, even but these protein exist, still have a considerable amount of patients that endocrine therapy is demonstrated former or acquired resistance, and predict that whether specific patient tumors will produce the ability of replying to endocrine therapy and be still weak.

Having evaluation with the gene of the similar expression pattern of ESR1 in the mammary cancer biopsy provides and has increased the method that the ESR1 predictive is worth.And the key molecule mechanism major part that relates in the mammary cancer is unknown.For the illustrating and can be used for the treatment of the patient with breast cancer or have for the patient's that mammary cancer causes danger the exploitation of new treatment target of the molecule mechanism of the exploitation of the biomarker of the hormone response that is used for mammary cancer, mammary cancer, identify regulated by ER or with the gene of ER coexpression be very important.

In addition, identify that at present the main mode that mammary cancer exists is the detection that fine and close tumor tissues is existed.Can finish this detection by the direct inspection of breast outside or by mammography or other x-ray imaging method, this detection has different successful degree (referring to, Jatoi, Am.J.Surg., 177 volumes, 518-524 page or leaf, (1999)).In order to determine that specific tumors is the ESR1 male, be necessary to obtain to be used for the tumor biopsy tissue sample that IHC analyzes.This method is expensive and have invasive, and will make the patient be exposed to complication as infecting.Very expectation can be carried out less invasive diagnositc analysis on blood, because always can not obtain the tumor tissues that is used to analyze.

Therefore, whether the tumour that needs more special and still less invasive method to be used to detect the patient is the ESR1 male.In addition, the method that more needs to provide the tumour of determining particular patient---no matter whether to have ER---will how to reply endocrine therapy.This will make the doctor can make the decision of wiser relevant treatment selection scheme, and can give patient's prognosis more accurately.In addition, need be used to identify the method for following compound, this compound will improve the response rate of breast cancer tumour to endocrine therapy.

Summary of the invention

As described in after this, the present invention be tested and appraised regulated by ER or with a large amount of genes of ER coexpression, overcome the defective in the methods availalbe of hormone response of the positive mammary cancer of present detection ER.For example have as the surrogate markers thing of hormone response with as the purposes of the potential treatment target of mammary cancer specificity corresponding to the mRNA transcript of these genes and protein.

And the present invention has identified endocrine therapy (is being comprised with aromatase inhibitor letrozole (Femara ^TM) treatment) produce and reply and the gene that does not produce differential expression in the breast cancer tumour of replying.

The present invention has identified several genes of expressing relevant encoding secreted protein with ESR1, and these secreted proteins comprise: TFF1; The trifolium factor 3 (TFF3); Serine or cystatin clade A member 3 (SERPINA3); Prolactin inductive protein (PIP), matrix Gla protein (MGP); The III receptor (TGFRB3) of transforming growth factor-beta; With contain zinc α-2-glycoprotein 1 (AZGP1).These protein can form the basis based on the predictive biomarker of serum.Listed the protein accession number of all genes in the various embodiments of the present invention, identified and their single-gene bunch number, gene symbol and their marking protein in the table 6.

Detailed Description Of The Invention

The present invention relates to the evaluation of gene, described gene regulated by ER or with the ER coexpression.No disease interval of replying with internal secretion, growing and the relevant tumor phenotypes of growing of whole survival time have been determined in the expression of ESR1 in the primary breast cancer.In big mammary cancer sample, found to have statistics significant correlation highly between ESR1 genetic expression and 18 kinds of other genetic expressions.In view of the coexpression of these genes in the breast cancer cell and ER gene, these genes and their expression product can be used for the breast cancer relapse patient or be in the patient or the patient with breast cancer's of mammary cancer initiation potential processing, prognosis and treatment.These genes have been shown in the table 1.Can obtain the complete sequence of disclosed these 18 kinds of genes and all other genes among the application with bunch accession number of the single-gene shown in the table 6.

The method that detects the mRNA expression level is well known in the art, includes but not limited to the Northern blotting, reverse transcription PCR, real-time quantitative PCR and other hybridizing method.

For detecting available from a large amount of openly levels of the mRNA transcript of gene, a useful especially method relates to mark mRNA and the hybridization of oligonucleotide oldered array.This method allows to detect simultaneously the transcriptional level of this several genes to produce gene expression profile or pattern.In another embodiment, derive from object sample gene expression profile can with the gene expression profile of the sample that derives from the individuality that does not have this disease relatively, determine whether thus that described object suffers from or dangerously suffer from mammary cancer.

Strong correlation between ER generegulation and this 18 kinds of generegulation has been supported these genes and ER gene to regulate altogether and has been this hypothesis of the biomarker of functional ER transcription (transcriptosome) therefore.Having 10 kinds of genes (gene 8-17) to be proved with the ER gene-correlation or by oestrogenic hormon in the gene of listing in the table 1 directly regulates.Preceding 7 kinds of genes (gene 1-7, promptly non-voltage-gated sodium channel 1 α (SCNN1A) shown in the table 1; SERPINA3; N-acyl sphingosine hydroamidase (ASAH); Lipocalin 1 (LCN1); TGFBR3; The IIB subtribe of glutamate receptor precursor 2 (GRIA2) and Cytochrome P450 (phenylethyl barbituric acid inductive CYP2B)) never be proved relevant before with the expression of ER in the mammary cancer.

Therefore, the invention provides the multiple gene of regulating with ER in big mammary cancer sample.Can select at least one mark of ER as an alternative in these genes arbitrarily.In useful especially embodiment, can select multiple in these genes, and the mRNA that monitors them simultaneously expresses to provide with the express spectra in all fields.

In another embodiment, can include but not limited to the level of gene expression product (protein) in blood, blood plasma, serum, lymph, CSF, gall-bladder liquid, ascites, urine, ight soil and the bile at various body fluid.Can be as there be the replacement mark of ER in this expression product level on the tumour cell, and the index that can provide the endocrine therapy of patient tumors to reply.

In addition, one or more expression of gene spectrums can provide valuable molecular tool in these genes, are used for detecting mammary cancer internal secretion molecular basis of replying and the breast cancer treatment effectiveness that is used to assess medicine.When cellular exposure in various modification conditions, when contacting with medicine or other bioactive molecule, express spectra can be used to indicate these effects with respect to the change of baseline spectrum.

In another embodiment, the present invention identifies and endocrine therapy is being produced and do not producing the gene of expressing with different levels in the breast cancer tumour of replying.Because the differential expression of these genes can utilize these genes and/or their expression product whether will make the determinacy of favourable prediction of replying to endocrine therapy to increase relevant particular patient breast tumor.These genes are that (neuro-oncological ventral antigenl NOVA1) with heavy chain immunoglobulin γ 3 constant regions (IGHG3), has listed these genes to neural tumor veutro antigen 1 in table 2.Can detect these open expression of gene levels by the protein of measuring pairing mRNA of genetic expression or genes encoding.Can be in office what body fluid easily, include but not limited to measure protein in blood, blood plasma, serum, lymph, CSF, gall-bladder liquid, ascites, urine, ight soil and the bile.

Whether therefore, the invention provides the cell that is used for definite specific mammary cancer sample has internal secretion to reply the method for phenotype.Term used herein " internal secretion is replied " means growth or the hyperplasia that can slow down or stop breast tumor or cancer by following treatment, and this treatment causes change on tumour cell, the ER activation that promptly increases or reduce.

Term used herein " endocrine therapy " means the treatment that can cause any kind that the ER activatory increases or reduces on the tumour cell as the main aspect of its clinical effectiveness directly or indirectly.Therefore, the term endocrine therapy includes but not limited to seal the medicine of ER and as the medicine of mixing agonist-antagonist of ER and reduce the treatment of endogenous estrogen concentrations, includes but not limited to, for example aromatase inhibitor, progesterone and LHRH.

Therefore, the invention provides the screening patient with breast cancer with determine patient's breast tumor to endocrine therapy produces the method for the possibility of replying, the breast cancer treatment of authentication method, the monitoring certain drug of useful medicament is renderd a service in the treatment patient with breast cancer method and in the breast cancer tumour cell the special carrier that duplicates.

Be used in letrozole (FEMARA ^TM ) to the definition of the objective response in the Tamoxifen comparative studies

Measurable disease

1. reply (CR) fully: by the disappearance of all definite known diseases of 2 observations that are no less than 4 weekly intervals.

2. part is replied (PR): be the effectiveness of determining treatment, by being no less than reducing of 50% or above total tumour lesion size that 2 times of 4 weekly intervals observe to measure.In addition, new damage or any damage progress can not appear.

3. do not change (NC): can not be defined as reducing of 50% total tumor size, not show that one or more sizes of measuring damage have 25% increase yet.

4. PD (PD): one or more sizes of measuring damage have 25% or above increase or new damage occurs.

The clinical response assessment

Main efficacy variables be utilize The World Health Organization's standard (referring to, report cancer therapy result's WHO handbook (WHO Handbook for Reporting Results of CancerTreatment)), by the tumor response of clinical examination assessment.It is defined as the percentage that has the patient of CR or PR in each treatment group, wherein in the time of 4 months, passes through clinical definite CR of chest palpation and PR.Possible replying is CR, PR, NC, PD maybe can not assess/can not estimate (NA/NE).Palpable homonymy axillary lymph knot involves the tumour grade that reduces clinical CR.In addition, also considered other factors, preserved the patient's of operation (quadrant surgical blanking/lumpectomy) rather than mastectomy percentage as the experience breast.Become inoperable patient or 4 months the time still inoperable patient can be regarded as the treatment failure.

Be used for detecting the method for the gene that mammary cancer and ESR1 regulate altogether

Material and method

Cell cultures

Be added with 0.03mg/mL endothelial cell growth feed supplement (ECGS), and growth U373 cell among the DMEM/F-12 of 0.1mg/mL heparin and 1xPen/Strep (ATCC, Rockville, MD).Cell grows into about 40% and is paved with, and washes once with nutrient solution then.Then with substratum or substratum+PDGF20ng/mL culturing cell 48 hours.Have 5%FBS, 0.03mg/mLECGS, (ATCC, Rockville MD) are paved with to about 40% growth people venous endothelial cell HUVEC in the F-12 substratum of 0.1mg/mL heparin and 1x Pen/Strep, wash once with nutrient solution then.Grown cell is 48 hours in substratum or substratum+VEGF 50ng/mL.At the MEM+2mM L-glutaminate, 0.1mM NEAA, the 1mM Sodium.alpha.-ketopropionate, the 0.1mM Sigma I8405, (ATCC, Rockville MD) are paved with to 80% growth breast cancer cell line MCF7 among the 10%BSA.Wash all cell cultures 2 times with ice-cooled PBS, scrape from culture dish then, in refrigerative PBS, precipitate, quick freezing in liquid nitrogen.

Specimen preparation

From 21 RNA samples of No. 14 core needle biopsy tissue extraction, wherein before new auxiliary endocrine therapy begins, from participating in III phase letrozole (FEMARA at random ^TM, Novartis Pharma, BasalSwitzerland) patient to the Tamoxifen test collects described biopsy, and wherein said test is unsuitable for the postmenopausal women that breast is preserved former aggressive mammary cancer of operation at suffering from.

Utilize Trizol (Life Technologies, Gaithersburg, MD) primary breast cancer of collecting from other 30 Sweden, 1 other ESR1+ breast tumor surgical operation biopsy, 2 HUVEC samples, 2 from extracting RNA the sample of glioblastoma clone U373-MG and a MCF7 sample.After the agreement acquisition informed consent according to the approval of local Ethics Committee, collect clinical sample.Buy 2 RNA samples, one for soak into III phase duct carcinoma (Ambion, Austin, TX), another be 2 normal galactophore tissues miscellany (Clontech, Palo Alto, CA).The total number of samples of preparation is 59, comprises 53 mammary cancer biopsies and 1 blended normal breast sample.As Lockhart etc., Nat.Biotechnol., 14 volumes, 1675-1680 page or leaf (1996) is described, total RNA QIAGEN RNEASY ^TM(CA) purifying is handled back and HUGENE to post for Qiagen, Valencia ^TMFL 6800 arrays (Affymetrix, Santa Clara, CA) hybridization.

The level cluster

Owing to calculate restriction, the subclass input of 1,156 gene of HuGeneFL 6800 arrays is used for cluster.This subclass is by GENECHIP ^Software (Affymetrix, Santa Clara, CA) genomic constitution of approval (call) existence, these genes are present at least one of 59 samples and 20 times differential expression are arranged, promptly at the normal mean difference (AvDif) that mixes between at least one of mammary tissue sample and these 59 samples.This gene subclass has been represented those genes that some level of difference are arranged in the ideal case between normal and tumour.This subclass has been got rid of those does not all have the gene of expressing or not having significant change at least one sample in any sample.Utilize GENESPRING ^TM3.2.8 (Silicon Genetics, Redwood City CA), carry out cluster with the genetic expression value to gene and sample, wherein stride sample the mean difference observed value of each gene is carried out normalization method so that intermediate value is one.Measure the genetic expression similarity by normalized correlation, minor increment be 0.001 and separation rate be 0.5.From collected the tabulation with the gene of ESR1 copolymerization class of the branch of the obtain tree derivation that contains the ESR1 gene.

The result

The laboratory sample tree

Not or have main bunch of collection of sample that very low ESR1 expresses, do not have sample bunch collection that high ESR1 expresses, but do not describe the clear branch (Fig. 2) of these two sample classes at the other end at the nearly end of tree derivation.The scope from-24.08 to 3501.6 of the AvDif value of ESR1, the normal breast displayed value is 124.Normal breast sample bunch collection is on the border that has low sample of expressing and the sample with high expression level for 18 kinds of genes of this paper report usually.Among Fig. 2, the mean value of the ESR1 AvDif of all samples of bunch collection on normal breast is 66.37, and standard deviation is 163.54.The mean value of the ESR1 AvDif of bunch all samples of collection below the normal breast sample is 1440, and standard deviation is 936.

Endothelium and glioblastoma cell cultures sample and their bunch collection of cell type separately are in the branch that is different from the tumor biopsy tissue.Endothelium and glioblastoma branch are positioned at the end of the tree derivation with low ESR1 expression.In cluster analysis, comprise clone, so that by providing the cell type that may reside in the breast tumor such as endothelium and epithelial cell and visibly different cell type such as glioblastoma to improve the cluster of gene.

Gene with ESR1 copolymerization class

18 kinds of genes and ESR1 copolymerization class (table 1).These genes have high expression level in the ESR1 positive and hang down the unique pattern (Fig. 2) of expressing in the ESR1 negative samples.With 7 kinds of genes of ESR1 copolymerization class, promptly SCNN1A, SERPINA3, ASAH, LCN1, TGFBR3, GRIA2 and CYP2B (table 1) never link together with oestrogenic hormon stimulation or mammary cancer in the past.

Be considered to the protein of estrogen regulating in the past with 6 kinds of genes of ESR1 copolymerization class, as the prediction or the prognosis biomarker of mammary cancer, these 6 kinds of genes are carcinomebryonic antigen relevant cell adhesion molecule 5 (CEACAM5), LIV-1 protein (LIV-1), PIP, MGP, TFF3 and TFF1 (being also referred to as PS2) (referring to table 1).

CEACAM5 is reported in the immunoreactivity glycoprotein of expressing in the 10-95% mammary cancer.In research to 298 routine breast tissue samples, find in the ESR1 positive/PGR-positive tumor the CEACAM5 protein level the highest (referring to, Molina etc., Anticancer Res., 19 volumes, 2557-2562 page or leaf (1999)).Except with outside the Pass ESR1 expresses mutually, at Zach etc., J.Clin Oncol, 17 volumes point out in the report of 2015-2019 page or leaf (1999) that CEACAM5 is relevant with mammary gland globin (mammaglobin) 1 (MGB1) expression.Point out also in the same report that the MGB1 level is relevant with the ER level, this has supported the gene cluster result.

LIV-1 is the ER gene of document write up.By ESR1 dependency mechanism, Urogastron (EGF), transforming growth factor-alpha (TGF α) and insulin-like growth factor 1 (IGF1) induce LIV-1 (referring to, EI-Tanani etc., J.Steroid Biochem.Mol.Biol., 60 volumes, 269-276 page or leaf, (1997)).

PIP perhaps is called total cystic disease liquid protein 15 (gross cystic disease fluidprotein 15), and it is induced by prolactin and male sex hormone.PIP expression level relevant with ESR-1 (referring to, Clark etc., Br.J.Cancer, 81 volumes, 1002-1008 page or leaf (1999)) with the PGR-positive.

MGP belongs to osteocalcin/matrix gla protein families, and is relevant with the organic substrate of bone and cartilage, and is considered to work as the inhibitor that bone forms.Oestrogenic hormon is the strong inductor of MGP genetic expression.

Oestrogenic hormon is also induced TTF1 and TTF3 consumingly.The trifolium factor is the stably excreting protein of expressing in gastrointestinal mucosa.They can play and protect mucous epithelium to avoid the effect that damages and help to cure.TFF3 can be the predictive biomarkers of endocrinotherapy for breast cancer.It oestrogenic hormon reply and in the unresponsive breast cancer cell line of oestrogenic hormon, do not express, and its can by the expression of control APC and E-cadherin-catenin mixture play the effect that promotes cell migration (referring to, Efstathiou etc., Proc.Natl.Acad.Sci.USA, 95 volumes, 3122-3127 page or leaf (1998)).As previously discussed, TFF1 sets up the predictive biomarkers that good endocrine therapy is replied, and it is reported, by estradiol rather than progesterone, dexamethasone or Standone can increase TFF1 mRNA level (referring to, Prud ' homme etc., DNA, 4 volumes, 11-21 page or leaf (1985)).And, it is reported, Tamoxifen suppress the estradiol of TFF1 induce (referring to, Prud ' homme, above-mentioned quoted passage).

With another gene of ESR1 copolymerization class, i.e. hepatocyte neclear factor 3 α (HNF3A) activation TFF1 (referring to, Beck etc., DNA Cell Biol., 18 volumes, 157-164 page or leaf (1999)).Proved HNF3A and ESR1 copolymerization class (Perou etc., Nature, 406 volumes, 747-752 page or leaf (2000)) in the express spectra of 65 breast tumor in the past.In the report of Perou etc. above, 3 other genes that table 1 is listed also with ESR1 copolymerization class: LIV-1; Hepsin (HPN) transmembrane protein enzyme, it has basic effect in cell growth and morphocytology are kept; X box conjugated protein 1 (XBP1), it is in conjunction with HLA-DR-α promotor, and can be in the B cell as transcription factor (referring to, Liou etc., Science, 247 volumes, 1581-1584 page or leaf (1990)).

AZGP1 with the gene of ESR1 copolymerization class in be unique, because before it not with internal secretion reply be related and be considered to the mammary cancer differentiation biochemical markers (referring to, Diez-ltza etc., Eur.J.Cancer, the 29A volume, 1256-1260 page or leaf (1993)).AZGP1 is the secreted protein that stimulates degradation of lipid in the adipocyte, and it can impel a large amount of loss of fat in the patient with advanced cancer.The extracellular domain of itself and I class MHC antigen α chain has high similarity.

Holistic approach to genetic expression on the mRNA level is the strong instrument of research complex biological knowledge topic as mammary cancer.Here, utilize the normalized correlation algorithm to carry out cluster analysis, thereby can identify the gene of regulating altogether with ESR1 expressing array data.Found 18 kinds of genes, comprised that the known ESR1 of being subjected to regulates or 11 kinds of relevant genes take place with breast cancer tumour.Enjoyably, 4 kinds of genes that exist in the ESR1 branch described herein, promptly LIV1, HPN, XBP1 and HNF3A are identified the member of chamber epithelium ESR1 gene cluster, and be described referring to (Nature, 406 volumes, 747-752 pages or leaves (2000)) such as Perou.XBP1 is also relevant with the ESR1 state in the report of the 3rd part of breast tumor gene expression profile of Bertucci etc. (Hum.Mol.Genet., 9 volumes, 2981-2991 page or leaf, (2000)).HPN, HNF3A and XBP1 and the copolymerization class of ESR1 show these genes as LIV1 by estrogen regulating, and should be considered to the possible mark of complete ER signal pathway.

This is the reported first of dependency between ER and following 7 kinds of genes: SCNN1A, SERPINA3, ASAH, LCN1, TGFBR3, GRIA2 and CYP2B.Gene TGFBR3 and LCN1 participate in cytodifferentiation and hyperplasia, downward modulation during and they at specific cells are---also being the clone of ESR1 sample on the origin---can cause tumour generation and ESR1 and these genes the copolymerization class (referring to, Bratt, Biochim.Biophys.Acta., 1482 volumes, 318-326 page or leaf (2000)).

Table 1 is presented in the level cluster of 1126 genes in 53 breast tumor biopsies, 1 normal breast and 5 the clone samples gene with ESR1 copolymerization class.At the shown GenBank accession number of each gene is the accession number of sequence, and from then on sequence can obtain to be used in the detection that 25mer probe on the Affymetrix gene chip is used for this gene.With+confirmed to have the gene of the expression positive relevant before representing with ER.

The gene of table 1 and ESR1 copolymerization class

The known dependency with ESR1 of gene GenBank accession number

1.???????SCNN1A???????????X76180????????????-

2.???????SERPINA3?????????X68733????????????-

3.???????ASAH?????????????U70063????????????-

4.???????LCN1?????????????L14927????????????-

5.???????TGFBR3???????????L07594????????????-

6.???????GRIA2????????????L20814????????????-

7.???????CYP2B????????????M29874????????????-

8.???????CEACAM5??????????M29540????????????+

9.???????MGB1?????????????U33147????????????+

10.??????LIV1?????????????U41060????????????+

11.??????PIP??????????????HG1763????????????+

12.??????MGP??????????????X53331????????????+

13.??????TFF3?????????????L08044????????????+

14.??????TFF1?????????????X52003????????????+

15.??????HNF3A????????????U39840????????????+

16.??????HPN??????????????X07732????????????+

17.??????XBP1?????????????M31627????????????+

18.??????AZGP1????????????X59766????????????-

19.??????ESR1?????????????X03635????????????+

The predictability mark that internal secretion is replied in the biopsy before the treatment

In the present invention is aspect another, 136 breast biopsy tissues have been obtained from 53 routine patients.Extract RNA from 116 biopsies.43 biopsies from 35 routine patients have been prepared express spectra.Identified the predictability mark that endocrine therapy is replied in the breast tumor.To derive from letrozole (FEMARA ^TM) before the treatment preparation the biopsy of express spectra and patient's clinical effectiveness broken down as follows: 4 routine patients have CR, and 9 routine patients have PR, and 4 routine patients have NC and 4 patients that PD is arranged.

For group with the Tamoxifen treatment, there is not the patient in the CR classification, 10 routine patients have PR, and 7 routine patients have NC, and 4 routine patients have PD.

Patient with CR or PR classifies as " respondent ", and the patient with NC or PD classifies as " non-responder ".From accepting letrozole (FEMARA ^TM) relatively 8,000 expression of gene between these two groups in the biopsy before patient's the treatment of administration.Numerical value (AvDiff) is illustrated in expression of gene level in the specific sample.Because the reason of calculating, with respect to all respondents, the mean value of the AvDiff value of each gene on the computing array.Then, each gene of each independent sample in these mean values and the non-responder's group is made comparisons.Identifying two has the gene of 3 times or higher differential expression at respondent's mean value and each non-responder's sample room, i.e. NOVA1 and IGHG3, and they are listed in table 2 and 6.Table 2 also comprises V5 biopsy only for reference (treatment back) data.

With FEMARA ^TMHave during the treatment NC or PD the women biopsy relatively, find that these two gene IGHG3 and NOVA1 are subsequently finally to FEMARA ^TMExpress with higher level in the women's that treating is positive replys the treatment pre-neoplastic.Use the Mann-Whitney rank test, for gene NOVA1, the diversity ratio of intermediate value is big by the desired value of probability (P=0.012) between two groups (comprising the V5 sample).For the IGHG3 gene, these data do not have the significance on the statistics.These genes (IGHG3 and NOVA1) do not have differential expression in the biopsy from Tamoxifen treatment patient, therefore can not provide mark to indicate whether and can produce favourable replying to Tamoxifen.

In order to determine to utilize following sign: NOVA1 (single-gene ID Hs.214) to be positioned on the karyomit(e) 14q by the NOVA1 gene uniquely, by mRNA accession number NM_002515 and protein accession number NP_002506 sign.

For IGHG3 gene (Hs.300697), this gene also is positioned on the karyomit(e) 14q, is identified by mRNA accession number BC016381.There is not the protein accession number.

There are several biological properties in gene IGHG3 and NOVA1, and these biological properties make these genes be suitable for use as diagnosis marker and/or treatment target.IGHG3 is relevant with heavy chain disease (HCD).HCD is the lymphocytic hyperplasia disease of natural generation, wherein has the variable fragment of mono-clonal Ig heavy chain (H) in serum or urine.NOVA1 is the nRNA conjugated protein with strict expression of regulating, and in the mouse that grows, this protein is limited in the neurone of CNS.In opsoclonus-ataxia (POA) patient of tool tumour formation sign, observed anti-this antigenic antibody.POA is an autoimmune disease, suffers from breast cancer in this disease or the abnormal motion control of eyes, trunk and four limbs takes place for the women of small cell lung cancer.Breast tumor is expressed the NOVA1 gene singularly in this disease.This causes the immunne response of the CNS that attacks natural expression NOVA1.Be used to the diagnosis of POA with the sero-reaction of NOVA1 fused protein, and show and have recessive mammary gland, gynaecology or lung tumor.

Table 2 and non-responder relatively reply (FEMARA before the patient treatment ^TM) have the gene of variable expression in the breast biopsy tissue

	The respondent													The non-responder
																						???????????????????????????????V0							??????????????????????????????V5						??????????????????V0				??????????????????V5
	PG sample No.	??P380- ??2f	p382f	p141f ▲	p610f	p615f	p611f	p580f	p387f	p592f	p143f ▲	p111- 2f	p582f	??p598f ??▲	??p568f ??▲	??p136- ??2f	??p609f	??p613f	??p391f	??p589f	??p566- ??2f																						??p570- ??2f▲
1GHG3																						??19845 ??P	260.5 P	682.1 P	1551 P	2607 P	1051 P	18 A	128.8 A	631.4 P	2050 P	2869 P	2424 P	??707.1 ??P	??630.1 ??P	??119.4 ??A	??532.9 ??P	??491.6 ??P	??1451 ??P	??1974 ??P	??2833 ??P	??5351 ??P
	NOVA1	??118.5 ??A	325.2 P	33.9 P	158.5 P	250.8 P	130.5 P	730 P	377.6 P	395.8 P	24.9 A	94.2 P	431.1 P	??20.8 ??A	??36.9 ??A	??7.1 ??A	??51.3 ??P	??51.3 ??P	??13.4 ??A	??87.4 ??P	??57.6 ??P																						??27.2 ??A

Patient's identifier of PG sample number=uniqueness

V0=investigates the biopsy (before the treatment) of sampling for the first time

The 5th investigation of V5=(treatment back)

▲=find to exist ER based on gene expression profile and ICH

This expression of gene level in numerical value (AvDiff)=specific sample

Absolute approval (AbsCall)=determine by Affymetrix software whether gene expresses in the sample, and with A (shortage); M (critical); Or P (existence) expression.

Predictability mark from treatment back biopsy

In another aspect of this invention, identified from the answer logo for the treatment of the back patient.For this purpose, will be from letrozole (FEMARA ^TM) treatment patient biopsy, from the sample of V5, promptly the treatment after biopsy be divided into two kinds---respondent and non-responder.Biopsy from the patient with CR or PR is used as the respondent, is divided into the non-responder from the biopsy of the patient with NC or PD.Because the reason of calculating, at the V5 respondent, the mean value of the AvgDiff value of each gene on the computing array.Then, each gene of each independent sample in these mean values and the non-responder's group is made comparisons.Identify by 7 kinds of genes of 8 groups of probes representative and differential expression (table 3) more than 3 times is arranged at each sample room of respondent's mean value and non-responder's group.Table 3 also comprises the data from biopsy V0 before the treatment only for reference.Two groups of different probes that are used for β-oxyphorase show, to FEMARA ^TMThe patient's who replys biopsy and non-responder's biopsy relatively have this higher genetic expression.Enjoyably, 2 genes of evaluation promptly 2 of HPN and the PIP positive and negative biopsy of ER at the ER that undertaken by genetic expression are tieed up in the level clusters and ESR1 copolymerization class.Use the Mann-Whitney rank test, for HPN (P=0.046) and lactotransferrin (P=＜0.001), intermediate value has the significant difference on the statistics between respondent and non-responder.In order to carry out the Mann-Whitney rank test, utilized all biopsy data, comprise V0 and V5 biopsy.

The mark tabulation comprises HPN and PIP.In the level cluster analysis, also find these genes and ESR1 copolymerization class.Independently analyze according to two, should think that HPN and PIP can be used for prediction to letrozole (FEMARA ^TM) the biomarker of function ER transcription of responsiveness.

Of Kazama (J.Biol.Chem., 270 volumes, 66-72 page or leaf, (1995)), HPN is the relevant serine protease of II class film, has confirmed that it activates human factor VII, and starts the blood coagulation approach and cause forming zymoplasm on cell surface.As (Proc.Natl.Acad.Sci.USA such as Torres-Rosada, 90 volumes, 7181-7185 page or leaf (1993)) described, it is believed that a large amount of neoplastic cells can be by this and other pathway activation blood coagulation system, cause thrombosis in hypercoagulability and the blood vessel, and hepsin works in their cell growth.As Tsuji etc., J.Biol.Chem., 266 volumes, 16948-16953 page or leaf (1991) is described, and the HPN expression of gene is height-limited; Promptly this gene is low expression level in most of bodily tissues medium level is expressed except high level expression in liver with in kidney.

Be reported in high level expression HPN in several cancerous cell lines and the ovarian cancer (nearest report, referring to for example Tanimoto etc., Cancer Res., 57 volumes, 2884-2887 page or leaf (1997)).In addition, although in liver the HPN high expression level, all ruined knock-out mice of two copies of HPN gene does not show the unusual and dysfunction of liver.WU etc. for example, J.Clin.Invest., 101 volumes, described in the 321-6 page or leaf (1998), in fact, these mouse do not show any recognizable phenotype.For example above-mentioned quoted passage Torres-Rosada etc. are described, confirmed the growth that the antibody of target HPN extracellular domain can block the liver cancer cell of overexpression HPN.

Identify two probes that are used for the β oxyphorase.This shows that after treatment β oxyphorase expression level in the respondent than in the non-responder is higher in (V5) tumour.The tumour relatively poor with vascularization compares letrozole (FEMARA ^TM) more successfully target vascular therapy form good breast tumor, and β oxyphorase expression level is relevant with the degree that these biopsy medium vesselses form.Lactotransferrin (LTF) is also included within the potential mark tabulation.LTF is the iron-binding protein matter of expressing in milk, and it is also expressed in the secondary granule of neutrophilic granulocyte.LTF participates in transportation storage and the chelating and the host defense mechanism of iron.It is reported, in about 50% breast tumor of measuring, lack LTF (referring to, Perou etc., Nature, 406 volumes, 747-752 page or leaf, (2000)).

Table 3 with to FEMARA ^TMTreatment produces the negative patient who replys and compares, and is right in tumour

FEMARA ^TMThe gene of finding among the patient of positive response with the higher level expression

??1	Hepsin transmembrane serine protease 1
		??2	The β oxyphorase
??3	The β oxyphorase
		??4	The ionic glutamate receptor, AMPA2
??5	The d1 of tumour differential expression

Table 4 with to FEMARA ^TMTreatment produces the negative patient who replys and compares, and is right in tumour

FEMARA ^TMThe gene of finding among the patient of positive response with the lower level expression

??1	Lactotransferrin
		??2	Prolactin inductive protein (PIP) a
??3	Sorbito dehy drogenase

Therefore, can be by any reliable method, include but not limited to method disclosed herein, in replying the patient of Femara and to Femara, do not produce among the patient who replys the absolute expression levels of measuring these genes or their gene product, then to this result and in unknown individual the expression level of same gene or gene product compare to determine whether unknown tumour will (comprise letrozole (FEMARA to endocrine therapy ^TM) treatment) produce and reply.

</entry></row></tbody></tgroup></table></tables>

Table 6 (is had to the Mrna sequence) except IGHG3 and PIP, at the single-gene of the complete genome group sequence of disclosed all genes among the application bunch number

This table also has the proteinic protein accession number of HUGO gene symbol and this genetic expression

GenBanK accession number protein

Gene (is used to design single-gene bunch number gene symbol

The Affymetrix probe) accession number

Non-voltage-gated sodium channel 1 α X76180 Hs.2794 SCNN1A prf:2015190A

Serine or cystatin member 3 X68733 Hs.234726 SERPINA NA

3

N-acyl sphingosine acid amides U70063 Hs.75811 ASAH sp:Q13510

Lytic enzyme (acid ceramidase)

Lipocalinl???????????????????????????L14927????????????Hs.2099??????LCN1???????prf：1908211A

The III receptor L07594 Hs.79059 TGFBR3 sp:Q03167 of transforming growth factor-beta

Glutamate receptor precursor 2 L20814 Hs.89582 GRIA2 pir:158181

Phenylethyl barbituric acid inductive Cytochrome P450-IIB M29874 Hs.1360 CYP2B pir:A32969

Carcinoembryonic antigen mRNA M29540 Hs.220529 CEACAM5 pir:A36319

Mammary gland globin 1 U33147 Hs.46452 MGB1 sp:Q13296-

The LIV-1 protein U41060 Hs.79136 LIV-1 pir:G02273 of estrogen regulating

Prolactin inductive protein HG1763 Hs.99949 PIP pir:SQHUAC

Matrix Gla protein X53331 Hs.279009 MGP pir:GEHUM

The trifolium factor 3 L08044 Hs.82961 TFF3 sp:Q07654

The trifolium factor 1 X52003 Hs.1406 TFF1 pir:A26667

Hepatocyte neclear factor 3 α U39840 Hs.299867 HNF3A pir:S70357

Serine protease hepsin X07732 Hs.823 HPN pir:S00845

X box binding protein matter 1 M31627 Hs.149923 XBP1 sp:P17861

Zn-α 2-glycoprotein X59766 Hs.71 AZGP1 pdb:1ZAG

Estrogen receptor alpha X03635 Hs.1657 ESR1 pir:S64737

X box binding protein matter 1 M31627 Hs.149923 XBP1 sp:P17861

Nerve-tumour veutro antigen 1 U04840 Hs.214 NOVA1 pir:138489

Immunoglobulin (Ig) γ 3 CH (G3m mark) M87789 Hs.300697 IGHG3 NA

β HbM 25079 Hs.155376 HBB prf:1701384A

Ionic glutamate receptor L20814 Hs.89582 GRIA2 pir:158181

Lactotransferrin X53961 Hs.105938 LTF pir:TFHUL

Sorbito dehy drogenase L29008 Hs.878 SORD sp:Q00796

The d1 U49188 Hs.272168 TDE1 NA of tumour differential expression

Pharmacogenomics

Pharmacogenetics/genomics is determined heredity/genome factor that participation is replied the individuality of foreign compound or medicine.Can be individual with prevention or treatment patient mammary cancer with marker expression of the present invention being had stimulation or inhibiting medicament or conditioning agent give.This treatment and individual pharmacogenomics must be considered together.The metabolic difference of therapeutical agent may cause serious toxicity and treatment failure by dosage and the relation between haemoconcentration that changes the pharmacologically active medicine.Therefore, understand individual pharmacogenomics and help effectively to screen preventative or therapeutic medicament (for example medicine).Further can utilize this pharmacogenomics to determine dosage and the treatment plan that is fit to.Therefore, the expression level that can measure mark of the present invention in the individuality is used for the medicament that is fit to of individual treatment or prevention with screening.

Pharmacogenomics research is because individual Chinese traditional medicine is disposed and different pharmaceutical efficacies that cause of effect or toxic clinical noticeable change (referring to .Linder for example, Clin.Chem., 43 roll up, No. 2,254-266 page or leaf (1997)).Usually, can be divided into two types pharmacogenetic condition.Change medicine to the mode of action of health, be called " drug effect of change " as the hereditary condition of monofactorial inheritance.Change health to the mode of action of medicine, be called " drug metabolism of change " as the hereditary condition of monofactorial inheritance.These pharmacogenetic conditions can be used as rare defective or common polymorphism occurs.For example, glucose-6-phosphate dehydrogenase (G6PD) defective is common hereditary enzymopathy, and wherein the main clinical complication is haemolysis to occur after taking in oxidant drug (antimalarial drug, sulfanilamide (SN), anodyne, nitrofuran) back and edible broad bean.

As an exemplary, the activity of drug metabolism enzyme is the main determining factor of drug potency and duration.The discovery of the genetic polymorphism of drug metabolism enzyme (for example, N-acetyl-transferase 2 (NAT2) and cytochrome P 450 enzymes CYP2D6 and CYP2C19) has explained why some patients do not obtain the effect of drugs of expectation or show excessive drug reaction and serious the poisoning behind the medicine of taking standard security dosage.

These polymorphisms are expressed with two kinds of phenotypes in the crowd: extensive metabolizer (EM) and poor metabolizer (PM).The popularity degree difference of PM in the different population.For example, the gene of coding CYP2D6 is the height polymorphism, and has identified several sudden changes in PM, and these all sudden changes have all caused the shortage of functional CYP2D6.When the poor metabolizer of CYP2D6 and CYP2C19 accepted standard dose, they often stood excessive drug reaction and side effect.As the mediation of the morphine monomethyl ether metabolite morphine that forms by CYP2D6 analgesic effect proved, if metabolite is the active treatment part, PM will show not treat and not reply.Other extreme case is the so-called supper-fast metabolizer of standard dose not being replied.Recently, identified supper-fast metabolic molecular basis: because due to the CYP2D6 gene amplification.

Therefore, can measure the expression level of mark of the present invention in the individuality or functional level is used for individual treatment or prevention with selection the medicament that is fit to.In addition, pharmacogenetics research can be used for the polymorphic allelotrope of coding drug metabolism enzyme or medicine target is carried out gene type to predict individual drug responses phenotype.When this knowledge is applied to administration and drug screening, can avoid untoward reaction or treatment failure with the expression modulators for treatment patient of mark of the present invention, strengthen the efficient of treatment or prevention thus.

Proteomics

Normal cell and the transformant excretory protein that can analyze cultivation may enter valuable protein body fluid and possible in the methods of the invention by the cancer cells secretion to identify.Can separation of supernatant, and can simplify protein mixture with the MWT-CO strainer.Use tryptic digestion protein then.Can load the peptide that tryptic digestion produces then on kapillary HPLC post, these peptides are with separated in kapillary HPLC post, and the electron spray ionisation source by custom-made directly is eluted in the ion trap mass spectrometer afterwards.On whole gradient, the 4 kinds of ions the strongest (peptide) by wash-out on the coupled columns carry out fragmentation, and dynamically get rid of before those ion of fragmentation simultaneously, can obtain sequence data.In this method, the sequence data that can obtain repeatedly to scan is corresponding to about 50-200 kind different proteins in the sample.Utilize the correlation analysis instrument,, can identify protein in the supernatant liquor with respect to these data of database retrieval as MS-Tag.

Measuring method

Experimental technique of the present invention depends on the measurement of cellular component.Measured cellular component can be from any aspect of cytobiology state.They can wherein measure the RNA abundance from transcriptional state; From the translation state, wherein measure proteinic abundance; From active condition, wherein measure activity of proteins.Cell characteristic also can for example be measured the RNA abundance (genetic expression) of one or more protein actives together with other cellular component from the mixing aspect.This part has been described and has been used for measuring the illustrative methods that drug responses or approach are replied cellular component.The present invention also is suitable for other method of carrying out this measurement.

Preferably, in the present invention measure the transcriptional state of other cellular component.Can be by measuring transcriptional state to the hybridization technique (following sub-fraction is described) of nucleic acid or nucleic acid analogue probe array or by other gene expression technique (small portion is described subsequently).Measure howsoever, the result is the data that comprise the value of representing mRNA abundance and/or ratio, and (under the identical situation of RNA degradation rate) it reflects the DNA expression rate usually.

In the various alternate embodiments of the present invention, can measure the aspect or the mixing aspect of the biological condition (as translation state, active condition) of non-transcribed state.

In one aspect of the invention, As time goes on can be, promptly in each stage of galactophore disease, in body fluid or mammary tissue sample available from the patient, measurement is corresponding to table 1, the mRNA of at least a gene of identifying in 2,3 or 4 or the expression level of proteins encoded are with the existing of mammary cancer among the monitoring patient, progress or prognosis.Can provide valuable information corresponding to the expression level of mRNA that is accredited as the gene relevant or coded protein about breast cancer treatment or progress with whole prognosis.Can detect the mRNA and the protein expression level of answering by standard method as described below with this gene pairs.

In a useful especially embodiment, can detect simultaneously each galactophore disease under the stage among the patient mRNA expression level of multiple open gene to produce transcribing or express spectra of galactophore disease as time passes.For example, can obtain corresponding to the most mRNA transcript of these genes from patient's mammary gland cell at different time, and carry out hybrid (wherein said oligonucleotide probe and the complementation of expectation gene transcription thing) with the chip that contains oligonucleotide probe, with than of the expression of relatively large gene in each stage of mammary cancer.

On the other hand, the cell analysis based on open gene can be used for identifying the medicament that can be used in breast cancer treatment.This method comprises: body fluid or the mammary tissue sample of a) suffering from the patient of galactophore disease with the contact of candidate's medicament available from suspection; B) detect at least a expression of gene level of identifying in the table 1,2,3 or 4; And c) with sample under lacking candidate's medicament situation in the expression of gene level compare, wherein with respect to the expression level under the medicament shortage situation, it is useful in breast cancer treatment that medicament exists under the situation in the sample change of expression level will indicate this medicament.As described below, can be by measuring protein level corresponding to the mRNA level of this gene or this genes encoding to detect gene expression dose.

When being used for more two or more value, used here term " similar " means these values and differs in 10% each other.

Here used term " candidate's medicament " refers to change or to reduce any molecule corresponding to the protein level of the mRNA level of at least one open gene or this genes encoding.Candidate's medicament can be natural or the synthetic molecule, as protein or its fragment, antibody, micromolecular inhibitor, nucleic acid molecule, and for example antisense nucleotide, ribozyme, double-stranded RNA, organic and mineral compound or the like.

The cell-less measurement method also can be used to identify and can interact to change the active compound of protein or its binding partners with a kind of protein or protein bound companion of open genes encoding.Cell-less measurement also can be used for identifying the compound that can regulate coded protein and its binding partners (as the target peptide) interphase interaction.

In one embodiment, be used to identify that the cell-less measurement method of this compound comprises reaction mixture, this reaction mixture contains a kind of protein and test compound or test compound storehouse of open genes encoding, and contain or do not have a binding partners, biological example is learned the target peptide or the small molecules of inactivation.Therefore, the invention provides an example that is used for identifying in the acellular method of the useful medicament of breast cancer treatment, this method comprises protein or its functional fragment or protein bound companion is contacted with test compound or test compound storehouse, and detects the formation of mixture.In order to detect, can be with specific mark labelled protein, and with different mark mark test compounds or test compound storehouse.Then, can detect test compound and protein or its fragment or protein bound companion's interaction by measurement level of two kinds of marks behind incubation and rinse step.Two kinds of marks all exist expression that interaction is arranged.

By detect surface plasma resonance-a kind of optical phenomena-instant BIA (biomolecular interaction analysis, Pharmacia Biosensor (AB)) also can estimate intermolecular interaction.This detection is depended on the change of the macromolecular mass concentration of improving quality at the biospecific interface and is not needed tagged molecule.In a useful embodiment, can be at sensor surface, restraint test compound library on microfluidic chambers (micro-flow cell) wall for example.Then, the solution that contains protein, its functional fragment or protein bound companion is circulated continuously at sensor surface.The change of the resonance angle of indicating on the signal record shows interaction has taken place.In the BIA of Pharmacia technical manual, describe this technology in detail.

Another embodiment of cell-less measurement method comprises: the protein, protein bound companion and the test compound that a) mix at least a genes encoding are to form reaction mixture; And b) detection protein and protein bound companion's interaction under test compound existence or shortage situation.Compare with there not being the interaction under the test compound situation, test compound exists protein and the interactional sizable variation of binding partners (enhancement or restraining effect) under the situation to show that test compound is the potential agonist (stand-in or toughener) or the antagonist (inhibitor) of protein active.Measure that composition can mix simultaneously or with after test compound contacted protein for some time to reaction mixture interpolation binding partners.Produce the effectiveness that dose response curve can be estimated compound by the compound that utilizes various concentration.Also can under the situation that does not have test compound, carry out blank determination by the formation of mixture between quantitative protein and its binding partners.

With protein or its binding partners of the protein that can detect ground mark such as radio-labeling, fluorescent mark or enzyme labelling, by immunoassay or can detect the formation of mixture between protein and its binding partners by chromatographic detection.

In preferred embodiments, can fixing protein or its binding partners to help from the not complex form of protein and its binding partners isolated complex and to be beneficial to the automatization of mensuration.Can be at the container of any kind, for example microtiter plate is finished the compound of protein and its binding partners in micro-centrifuge tube and the test tube.In particularly preferred embodiments, protein can with another kind of protein, for example glutathione-S-transferase merges to form fused protein, this fused protein can be adsorbed on matrix, for example (Sigma Chemical, St.Louis on the glutathione agarose gel beads, MO), with mark, for example protein companion and the test compound with the 35S mark mixes then, and hatches being enough to form under the condition of mixture.Subsequently, the washing pearl to be removing unconjugated mark, and with the matrix immobilization, measures radio-labeling.

Another method for immobilizing protein on matrix comprises and utilizes vitamin H and streptavidin.For example, can utilize technique known with vitamin H NHS (N-hydroxy-succinamide) biotinylated protein matter, and be fixed in the hole of plate of streptavidin bag quilt.

The cell-less measurement method also can be used to identify and can and regulate the medicament of the activity of proteins of this genes encoding with the protein interaction of at least a genes encoding.In one embodiment, proteinic catalytic activity is hatched and measured to protein and test compound.In another embodiment, measure the binding affinity of protein by methods known in the art to target molecule.

The present invention also provides the method for suffering from or the dangerous individuality of suffering from galactophore disease prevents and treats.Before the characteristic symptoms that the administration of preventative medicament can occur in galactophore disease manifests, can prevent the development of galactophore disease like this or delay its process.Aspect the treatment of galactophore disease, and do not require and kill mammary gland cell (for example cancer cells) or induce it that necrocytosis takes place.The treatment of opposite realization galactophore disease is needed only to be to slow down tumor growth to a certain extent or make some abnormal cellss recover normal.The example of the healing potion that is fit to includes, but are not limited to antisense nucleotide, ribozyme, double-stranded RNA and antagonist, as hereinafter describing in detail.

Terminology used here " antisense " refers to and at least a openly a part of complementary nucleotide sequence of the rna expression product of gene." complementary " nucleotide sequence refers to the nucleotide sequence that can carry out base pairing according to the complementary rule of standard Watson-Crick.That is, purine will match to form guanine with pyrimidine: cytosine(Cyt) and VITAMIN B4: thymus pyrimidine (in DNA) or VITAMIN B4: the association of uridylic (in RNA).Other uncommon base, Trophicardyl for example, 5-methylcytosine, 6-methyladenine, xanthoglobulin and other base can be included in the hybridization sequences, and they do not disturb pairing.

In all embodiments, the mensuration of cellular component should be carried out in the mode that is relatively independent of Measuring Time.

The mensuration of transcriptional state

Preferably, by the mensuration of nucleic acid and oligonucleotide arrays hybridization carrying out transcriptional state, see the description of this trifle.Some other transcriptional state measuring method has been described in this trifle after a while.

The general introduction of transcript array

In preferred embodiments, the present invention has utilized " oligonucleotide arrays " (being also referred to as " microarray " here).Can utilize the transcriptional state in the microarray analysis cell, particularly measure the transcriptional state of cancer cells.

In one embodiment, the polynucleotide of the detectable label by will representing the mRNA transcript that exists in the cell cRNA of fluorescently-labeled cDNA of total cell mRNA synthetic or mark (for example, from) produce the transcript array with microarray hybridization.Microarray is the surface with site of a series of ordered arrangement, and described site is used for combining (for example hybridization) with the product of cell or the many genes of organism genome (preferred great majority or nearly all gene).Can prepare microarray in many ways, hereinafter describe wherein several method.In any case make, the microarray of generation has some common traits.Array is reproducible, allows to produce a plurality of copies of specific array, and is easy to mutual comparison between these copies.Preferably, microarray is less, usually less than 5cm ², and they are by making in conjunction with stable material under (for example, nucleic acid hybridization) condition.Specific binding site or unique one group of binding site are with the product of the individual gene of specific combination cell in the microarray.Although every kind of specific mRNA has more than one physical bond site (after this being called " site "), for reason clearly, following discussion will suppose to have only single site.In specific embodiments, utilize the position addressable array that contains fixed known array nucleic acid in each position.

Be appreciated that as preparation and cell RNA complementary cDNA, and under the hybridization conditions that is fit to during, will reflect the abundance of the mRNA of this genetic transcription in the cell with hybridization level corresponding to the array site of any specific gene with itself and microarray hybridization.For example, when with (for example the using fluorophore) cDNA of total cell mRNA complementary detectable label or cRNA and microarray hybridization, corresponding in the cell not the array site of open gene (described " corresponding to " can the specific combination gene product) will not have or (for example almost do not have signal, fluorescent signal), corresponding to the site of the dominant gene of mRNA of coding strong relatively signal will be arranged.

The preparation of microarray

Microarray is known in the art, and by a surface composition, the probe (for example cDNA, Mrna, cRNA, polypeptide and fragment thereof) corresponding to gene product can in known location hybridization or combination take place specifically on sequence on this surface.In one embodiment, microarray is array (being matrix), each position is the discrete binding site of gene encoding production (for example protein or RNA) therein, and has the binding site at great majority or nearly all gene product in the organism genome.In preferred embodiments, " binding site " (after this claim " site ") is nucleic acid or the nucleic acid analog that specific connection cDNA or cRNA can specific hybridizations.The nucleic acid of binding site or analogue can be for example synthetic oligomer, full-length cDNA, less than the cDNA or the gene fragment of total length.

Although in preferred embodiments, microarray contains in the target organism genome binding site of all or nearly all gene product, and is this comprehensive not necessarily essential.Microarray can only have the binding site of part target organism gene.Yet usually, microarray has corresponding at least about 50%, and is more common at least about 85% usually at least about 75%, even more common more than about 90% and the most common binding site at least about 99% genomic gene.Preferably, microarray has the binding site of following gene, and described gene is relevant with checking purpose biology network model with test." gene " is defined as preferably having at least 50,75 or 99 amino acid whose open reading frame (ORF), will transcribe messenger RNA(mRNA) from this open reading frame in the organism (for example, if unicellular) or in some cells at multicellular organisms.Can be from the mRNA number of organism expressing or by the number gene from genomic detailed sign part extrapolation estimation genome.Behind the gene order-checking to organism interested, can determine the ORF number and identify the mRNA coding region by dna sequence analysis.The for example complete yeast saccharomyces cerevisiae that checked order (Saccharomyces cerevisiae) genome it is reported that it is had an appointment 6275 to be longer than 99 amino acid whose ORF.To the analysis revealed of these ORF have 5885 may the regulation protein ORF (referring to, Goffeau etc. for example, " life entity " with 6000 genes, Science, 274 volumes, 546-567 page or leaf, (1996), for all purposes, it is reference that integral body is incorporated this piece document into).Compare, the estimation human genome contains has an appointment 25,000-35,000 gene.

Preparation is used for the nucleic acid of microarray

As mentioned above, with " binding site " of specific connection cDNA specific hybridization normally attached to the nucleic acid or the nucleic acid analog in this site.In one embodiment, the binding site of microarray is the DNA polynucleotide corresponding at least one part of the genomic every kind of gene of organism.Can (for example increase by for example polymerase chain reaction (PCR) from genomic dna, cDNA, pass through RT-PCR) or the gene fragment of cloned sequence obtain these DNA, perhaps can utilize for example photolithography technology de novo synthesis sequence at chip surface, for example Affymetrix utilizes this different technologies directly to synthesize their oligomer on chip.Can select the PCR primer according to the known array of gene or cDNA, so that the unique fragment of amplification (that is, this fragment and microarray take up an official post its fragment what do not have the consecutive identical sequence of 10 above bases).Computer program (referring to, Oligo pl 5.0 versions (National Biosciences) for example) of great use in primer design with required specificity and the suitableeest amplification characteristic.Under the situation of binding site, expect near the amplification gene 3 ' fragment terminal sometimes, like this when the cDNA probe of oligomerization-dT initiation and microarray hybridization, less than the probe combination effectively of total length corresponding to very long gene.Usually, the length of each gene fragment is between about 20bp and about 2000bp on the microarray, and more generally between about 100bp and about 1000bp, about usually 300bp is to about 800bp.PCR method is known, for example editors such as Innis, " PCR Protocols:A Guide to Methods and Applications ", AcademicPress Inc., San Diego, CA has described this method in (1990), and for all purposes, it is reference that integral body is incorporated this piece document into.Clearly, computer-controlled robot system is useful for separation and amplification of nucleic acid.

The alternative approach that generation is used for the nucleic acid of microarray is for example to utilize N-phosphoric acid ester or phosphoramidite chemosynthesis polynucleotide or oligonucleotide (Froehler etc., Nucleic Acid Res., 14 volumes, 5399-5407 page or leaf, (1986); McBride etc., Tetrahedron Lett., 24 volumes, 245-248 page or leaf, (1983)).Composition sequence length arrives between about 500 bases about 15, more generally arrives between about 50 bases about 20.In some embodiments, synthetic nucleic acid comprises non-natural base, for example Trophicardyl.As mentioned above, nucleic acid analog can be as the binding site of hybridization.The example of a suitable nucleic acid analog be peptide nucleic acid(PNA) (referring to, Egholm etc. for example, " PNA follows Watson-Crick hydrogen bonding rule and complementary oligonucleotide hybridization ", Nature, 365 volumes, 566-568 page or leaf (1993); Also referring to U.S. Patent number 5,539,083).

In alternate embodiment; in conjunction with (hybridization) site from the plasmid of gene, cDNA (for example expressed sequence tag) or phage clone or from wherein inset preparation (Nguyen etc.; " by with the quantitative hybridization analysis mouse thymus of cDNA clone array in differential gene expression "; Genomics; 29 volumes; the 207-209 page or leaf, (1995)).In a further embodiment, the polynucleotide of binding site are RNA.

Nucleic acid is attached to solid phase surface

Nucleic acid or analogue are incorporated on the solid support, and this solid support can be made by glass, plastics (for example polypropylene, nylon), polyacrylamide, nitrocotton or other material.The method that preferably nucleic acid is attached to the surface is to print on sheet glass, usually can be referring to Schena etc., and " with cDNA microarray Quantitative Monitoring gene expression pattern ", Science, 270 volumes, 467-470 page or leaf, (1995).This method is particularly useful for preparing the cDNA microarray.Also referring to DeRisi etc., " using the gene expression pattern in the cDNA microarray analysis human cancer ", Nature Genetics, 14 volumes, 457-460 page or leaf, (1996); Shalon etc., " using the dna microarray system of Two Colour Fluorescence probe hybridization Analysis of Complex DNA sample ", Genome Res., 6 volumes, 639-645 page or leaf, (1996); With Schena etc., " parallel Human genome group analysis; The expression based on microarray of 1000 genes ", Proc.Natl.Acad.Sci.USA, 93 volumes, 10539-11286 page or leaf, (1995)).For all purposes, integral body is incorporated every piece of aforesaid article into as a reference.

Second kind of method that preferably prepares microarray is the highdensity oligonucleotide arrays of preparation.Utilization is used for original position synthetic photolithography technology, and to produce the technology of the array contain thousands of oligonucleotide be known, wherein said oligonucleotide be positioned at surperficial prescribed position and with the complementation of regulation sequence (referring to Fodor etc., " the parallel chemosynthesis of space addressable that light instructs ", Science, 251 volumes, 767-773 page or leaf (1991); Pease etc., " the photoconduction oligonucleotide arrays that is used for the rapid DNA sequential analysis ", Proc.Natl.Acad.Sci.USA, 91 volumes, 5022-5026 page or leaf, (1994); Lockhart etc., " by expressing ", Nature Biotech., 14 volumes, 1675 pages (1996) with high density oligonucleotide array hybridization monitoring; U.S. Patent number 5,578,832; 5,556,752 and 5,510,270, for all purposes, it is reference that integral body is incorporated every piece of document into); The technology that is used for other method of synthetic fast and precipitation regulation oligonucleotide also is known (Blanchard etc., " high density oligonucleotide array ", Biosensors﹠amp; Bioelectronics, 11 volumes, 687-690 page or leaf, (1996)).When utilizing these methods, directly synthesize the oligonucleotide (for example 25 aggressiveness) of known array on as the deutero-slide glass on the surface.Usually, the array of generation is redundant, and each RNA is corresponding to several oligonucleotide molecules.Can select oligonucleotide probe to detect alternatively spliced mRNA.

Also can utilize other method for preparing microarray, for example masking method (referring to, Maskos and Southern, Nuc.Acids Res., 20 the volume, 1679-1684 page or leaf, (1992)).Although as recognized by those skilled in the art, preferred very little array, because it is less to hybridize volume like this, but in principle, can utilize the array of any kind, for example the dot blotting on the nylon Hybond membrane (referring to, Sambrook etc., " Molecular Cloning--A Laboratory Manual (second edition) ", 1-3 volume, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989), for all purposes, it is reference that integral body is incorporated this piece document into).

Produce label probe

Prepare total and poly (A) ⁺The method of RNA is known, has summarized this method in above-mentioned quoted passages such as Sambrook.In one embodiment, utilize guanidine thiocyanate cracking all kinds purpose cell in the present invention, then by the centrifugal extraction of CsCl RNA (Chirgwin etc., Biochemistry, 18 volumes, 5294-5299 page or leaf, (1979)).By using oligomerization-dT cellulose selective poly (A) ⁺RNA (referring to, Sambrook etc., last quoted passage).The purpose cell comprises wild-type cell, be exposed to medicine wild-type cell, have modification/cell of interferential cellular component and have modification/cell that is exposed to medicine of interferential cellular component.

By oligomerization dT primer or random primer reverse transcription from mRNA or alternatively directly prepare the cDNA of mark from RNA, described two kinds of technology all be known in the art (referring to, for example Klug and Berger, Methods Enzymol., 152 volumes, 316-325 page or leaf, (1987)).Can put together the dNTP of detectable label, carry out reverse transcription under the situation that most preferably fluorescently-labeled dNTP exists.Alternatively, can be under the situation that the dNTP of mark exists, by the in-vitro transcription of double-stranded cDNA synthetic with isolating mRNA be converted into mark sense-rna (referring to, Lockhart etc., " by expressing " with high density oligonucleotide array hybridization monitoring, Nature Biotech., 14 volumes; 1675 pages, (1996), for all purposes, it is reference that integral body is incorporated this piece document into).In alternative embodiment, lack synthetic cDNA or rna probe under the situation of detectable label, and coming this cDNA of mark or rna probe by streptavidin (streptavidin of for example puting together phycoerythrin) or the equivalent of for example introducing biotinylated dNTP or rNTP or some similar approach (the psoralene derivative photo-crosslinking that for example makes vitamin H is to RNA) and then adding mark subsequently.

When using fluorescently-labeled probe, known have many suitable fluorophores, comprise fluorescein, Liz amine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, CY3.5, Cy5, Cy5.5, Cy7, FluorX (Amersham) and other fluorophore (referring to, Kricka for example, " nonisotopic dna probe technique ", Academic Press, San Diego, CA (1992)).Be understandable that, should select the paired fluorophore of different emmission spectrum so that easily distinguish them.

In another embodiment, utilized the marker of non-fluorescent label.For example, can utilize radio-labeling or have different emmission spectrum paired radio-labeling (referring to, Zhao etc., " the High Density cDNA filter membrane is analyzed: the novel method of extensive quantitative analysis genetic expression ", Gene, 156 volumes, 207 pages (1995); Pietu etc., " by quantitatively hybridizing the preferential new genetic transcription thing of expressing in human muscle of announcement ", Genome Res., 6 volumes, 492 pages, (1996) with the High Density cDNA array).Yet because the scattering of radioactive particle and therefore need the binding site of wide interval, radioisotopic use is an embodiment preferred not too.

In one embodiment, by at 42 ℃, with ThermoScript II (as, ^TMII, LTI Inc.) hatches together and contain 0.5mM dGTP, 60 minutes cDNA of mixture of dATP and dCTP and 0.1mM dTTP and fluorescence deoxyribonucleotide (for example, 0.1mM rhodamine 110 UTP (Perken Elmer Cetus) or 0.1mM Cy3 dUTP (Amersham)) with complex sign.

Hybridize to microarray

Select nucleic acid hybridization and wash conditions, so that probe " combination specifically " or " hybridization specifically " are to the specific array site, that is, making probe hybridize, form duplex with the sequence array site with complementary nucleic acid sequence or combine does not still hybridize with the site with incomplementarity nucleotide sequence.As used herein, if the short person of two polynucleotide sequences is less than or equals 25 bases and utilize the standard base pairing rules that both do not have mispairing, if perhaps the short person of two polynucleotide sequences is longer than 25 bases and mispairing between the two is no more than 5%, think one of them polynucleotide sequence and another polynucleotide sequence complementation so.Preferably, polynucleotide complementation (not having mispairing) fully.By comprising the hybridization analysis of negative control, whether can be easy to prove the specific cross condition will cause specific hybridization (referring to, Shalon etc. for example, last quoted passage and Chee etc., last quoted passage).

The suitableeest hybridization conditions depend on the length (for example oligomer is with respect to the polynucleotide with 200 above bases) of label probe and fixed polynucleotide or oligonucleotide and type (for example, RNA, DNA, PNA).At above quoted passages such as Sambrook with at Ausubei etc., " Current Protocolsin Molecular Biology ", Greene Publishing and Wiley-interscience, the general parameters of nucleic acid specificity (promptly tight) hybridization conditions has been described among the NY (1987), for all purposes, it is reference that integral body is incorporated these documents into.When utilizing the cDNA microarray of Schena etc., typical hybridization conditions is to add among the 0.2%SDS hybridization 4 hours at 65 ℃, 5xSSC, in 25 ℃, the lavation buffer solution (1xSSC adds 0.2%SDS) of low stringency, wash subsequently, then in 25 ℃, the lavation buffer solution (0.1xSSC adds 0.2%SDS) of high stringency, wash 10 minutes (referring to, Shena etc., Proc.Natl.Acad.Sci.USA, 93 volumes, 10614 pages, (1996)).

At for example Tijessen, " Hybridization With Nucleic Acid Probes ", ElsevierScience Publishers B.V. (1993) and Kricka, " Nonisotopic DNA ProbeTechniques ", Academic Press, San Diego, CA also provides useful hybridization conditions in (1992).

Signal detection and data analysis

When utilizing fluorescently-labeled probe, preferably, detect the fluorescent emission in each site on the transcript array by scanning confocal laser microscopy.In one embodiment, utilize the excitation line that is fit to that each fluorophore in used two fluorophores is scanned separately.Alternately, can utilize laser, then the emission light of analysis of fluorescence group to be specific to the wavelength illumination sample of used fluorophore.In preferred embodiments, with lasing fluorescence scanning imaging instrument scanning array with computer-controlled X-Y rank (stage) and micro objective.Can realize exciting in succession of fluorophore with multi-thread mixed gas laser, and detect with photomultiplier after separately launching light by wavelength.At Schena etc., GenomeRes., 6 volumes, the 639-645 page or leaf has been described fluorescence laser scanning device in (1996) and other reference of quoting here.Alternately, Ferguson etc., Nature Biotech., 14 volumes, the 1681-1684 page or leaf, the fibre bundle that (1996) are described can be used for monitoring simultaneously the mRNA abundance level on many sites.

Recording signal is also passed through computer in preferred embodiments, for example utilizes 12 modulus plates that signal is implemented to analyze.In one embodiment, with graphic package (for example, Hijaak Graphics Suite) remove the hot spot of scan image, use image lattice programanalysis image then, this program will produce the electrical form that has write down the average hybridization under each wavelength of each site.

Agilent Technologies GENEARRAY ^TMScanner is desk-top 488nM argon laser analyser.This laser can focus on the point less than 4 microns sizes.This tolerance range allows the probe array with little probe unit to 20 microns is scanned.Laser beam focuses on the probe array, the Nucleotide of fluorescence excitation mark.Utilize then at used dyestuff in analyzing and selected spectral filter is carried out scanning.Finish the scanning of orthogonal coordinate by the traveling probe array.The dye molecule that is combined in the hybridization sample will absorb laser radiation and emitting fluorescence.Scioptics calibration fluorescence makes it pass spectral filter to select wavelength.Be used for above the hole that intensity differentiates by the second lens focus light then.Detect by high quick photomultiplier (PMT) then.The PMT outward current is converted to voltage readings by analog to digital converter (ADC), and the data after the processing are returned computer with the form of the pictorial element (pixel) of the fluorescence intensity level of sample spot or current scanning.Along with the computer that carries out that scans is an image with data presentation.In addition, the fluorescence intensity level of all samples of expression sample express spectra is with the computer-readable format record.

If necessary, can test and determine to proofread and correct " cross-talk " (or overlapping) between two kinds of fluorescence channels.For any specific hybridization site on the transcript array, can calculate the emission ratios of two kinds of fluorescence.This ratio is independent of the absolute expression levels of associated gene, is useful but may be subjected to the remarkable gene of regulating of medicament administration, genetically deficient or any other detection incident for expression.

Preferably, except identify to disturb positive or negative, it also is favourable measuring the interferential amplitude.This can be undertaken by the conspicuous method of those skilled in the art.

When being used for more two or more value, term " similarly " refers to when using same units as used herein, and two values numerically differ in 20%, or more preferably in 10%.

Other transcriptional state measuring method

Can measure the transcriptional state of cell by other gene expression technique known in the art.Several such technology produce the restriction fragment storehouse with limited complicacy that is used for electrophoretic analysis, as in conjunction with the method for two restriction enzymes digestion and phasing primer (phasing primer) (referring to, European patent 0 534858 A1 that on September 24th, 1992 submitted to such as Zabeau for example), or with the method for the site screening restriction fragment of the most approaching regulation mRNA end (referring to, Proc.Natl.Acad.Sci.USA such as Prashar for example, 93 volumes, 659-663 page or leaf, (1996)).Other sampling cDNA storehouse, method statistic ground, as each the enough bases (for example 20-50 base) by a plurality of cDNA of order-checking to identify each cDNA, or the short label (for example 9-10 base) that on the known location with respect to the mRNA end of stipulating, produces by checking order (referring to, Velculescu for example, Science, 270 volumes, 484-487 page or leaf, (1995)) to identify the approach pattern.

The measurement of others

In the various embodiments of the present invention, can measure the biological condition aspect except transcriptional state, as the translation state, reply to obtain medicine and approach active condition or mixing aspect.The details of these embodiments has been described in this part.

The measurement of translation state

Can by can detect ground mark or the protein expression of the probe in detecting genes encoding of mark subsequently.Usually, this probe is the expressed proteinic antibody of identification.

Term as used herein " antibody " includes but not limited to polyclonal antibody, monoclonal antibody, humanization or chimeric antibody and is enough to the binding antibody fragment to proteinic biological functionality antibody fragment.

In order to produce a kind of proteinic antibody of open genes encoding, can be by injection polypeptide or the various host animals of its fragment immunity.These host animals can include, but are not limited to rabbit, mouse and rat etc.According to host species, can reply with various adjuvant enhancing immunity, these adjuvants include but not limited to freund's adjuvant (fully with incomplete), mineral coagulant such as aluminium hydroxide, the human adjuvant of surfactant such as lysolecithin, poly alcohol, polyanion, peptide, oil emulsion adjuvant, Yao Kong Wei hemocyanin, dinitrophenol(DNP) and potentially useful such as BCG (bacille Camette-Guerin) and spillikin bacillus (Corynebacterium parvum).

Polyclonal antibody is the heterogeneic antibody molecular group from the animal serum that utilizes antigen immune, and above-mentioned antigen is target gene product or its antigenicity functional deriv for example.Be to produce polyclonal antibody, can inject for example above-described host animal of immunity by proteins encoded or its part that above-mentioned adjuvant has been added in utilization.

Monoclonal antibody (mAb) is the predominating of antibody population at specific antigen, and it can obtain by utilizing any technology that allows the continuous cell line cultivated to produce antibody molecule.These technology include, but are not limited to Kohler and Milstein hybridoma technology (1975, the nature (Nature) 256:495-497; And U.S. Patent number 4,376,110), human B cell hybridoma technology (people such as Kosbor, 1983, immunology today (Immunology Today) 4:72; People such as Cole, 1983, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA) 80:2026-2030) and EBV hybridoma technology (people such as Cole, 1985, monoclonal antibody and cancer therapy (MonoclonalAntibodies and Cancer Therapy), Alan R.Liss company, 77-96).These antibody can be any immunoglobulin (Ig) kind, comprise IgG, IgM, IgE, IgA, IgD and their any subclass.The hybridoma that produces mAb of the present invention can be at external or culturing in vivo.The method that produces the high mAb that tires in the body is present preferred production methods.

In addition, also can use technology (people such as Morrison, 1984, newspaper (Proc.Natl.Acad.Sci.USA) 81:6851-6855 of institute of NAS of production " chimeric antibody "; People such as Neuberger, 1984, nature (Nature) 312:604-608; People such as Takeda, 1985, nature (Nature) 314:452-454), it carries out with the gene splicing with human antibody molecules of suitable biologic activity by the gene with the mouse antibodies molecule of the suitable antigen-specific of tool together.Chimeric antibody is that wherein distinct portions is from the molecule of different animal species, and for example those have from the variable or hypervariable region of mouse mAb and the antibody of human normal immunoglobulin constant region.

Alternatively, can adopt technology (U.S. Patent number 4,946,778 that produce single-chain antibody; Bird, 1988, science (Science) 242:423-426; People such as Huston, 1988, newspaper (Proc.Natl.Acad.Sci.USA) 85:5879-5883 of institute of NAS; And people such as Ward, 1989, nature (Nature) 334:544-546) produce the single-chain antibody of difference expression gene.Single-chain antibody can be by making heavy chain and light chain Fv district segment link to each other by the amino acid bridge and produce single chain polypeptide and form.

More preferably, the technology that is used for producing " humanized antibody " antibody of white matter, its segment or derivative of laying eggs next life.These technology are disclosed in U.S. Patent number 5,932, in 488,5,693,762,5,693,761,5,585,089,5,530,101,5,569,825,5,625,126,5,633,425,5,789,650,5,661,016 and 5,770,429.

The antibody fragment of identification specific epitopes can produce by technique known.For example, these segments include, but are not limited to: the F that produces by the pepsin digested antibody molecule (ab ') 2 segments and by reducing the Fab segment that F (ab ') 2 pulsating disulfide linkage generate.In addition, can make up the Fab expression library (people such as Huse, 1989, science (Science) is 246:1275-1281) to have the evaluation of the pulsating quickly and easily of the specific mono-clonal Fab of expectation.

Then can be by the expression degree of known protein matter in the immunoassay test sample of utilizing above-mentioned antibody.This immunoassay includes but not limited to dot blotting, the western trace, competitiveness and noncompetitive protein bound assay method, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, fluorescence activated cell sorting (FACS) and other are used the method with broadly described method and many commercializations always in science and patent documentation.

For ease of detecting, especially preferably use sandwich ELISA, there is multiple variation mode in this assay method.All modes include within the scope of the present invention.For example, in general forward is measured, unmarked antibody is fixed on the solid substrate, and specimen is contacted with the bonded molecule.Through suitable incubation time, this time forms enough antibody antigen binary complex bodys.At this moment, adding is used the second antibody of the reporter molecules mark that can induce detectable signal and is hatched again, and incubation time is the formation of antibody-antigen-traget antibody ternary complex enough.With any unreacting substance flush away, and by observation signal determine antigenic existence or by with contain the antigenic control sample of known quantity and relatively come quantitatively.The variation mode that forward is measured comprises to be measured simultaneously and oppositely measures, and sample and antibody add in the bonded antibody simultaneously in wherein measuring at the same time; And the antibody of mark at first mixes, hatches with sample to be tested and adds to then in the unlabelled surface bonding antibody in oppositely measuring.These technology are conventionally known to one of skill in the art, and obviously can carry out little change." sandwich assay method " in this use means all variation schemes that comprise based on these two basic site technology.For immunoassay of the present invention, only limiting factor is that the antibody of mark must be the proteinic specific antibody of destination gene expression.

The most normal reporter molecules that uses is as enzyme or contain fluorophore or the molecule of radionuclide in this mensuration.In the case of enzyme immunoassay, enzyme is coupled to second antibody, this realizes by glutaraldehyde or periodic acid usually.Yet, as what be easy to recognize, there are quite a large amount of different interconnection techniques, they are conventionally known to one of skill in the art.Normally used enzyme comprises horseradish peroxidase, glucose oxidase, beta-galactosidase enzymes and alkaline phosphatase or the like.For the substrate that uses with certain enzyme, be typically chosen in the substrate that can produce detectable color change after the hydrolytic action of corresponding enzyme.For example p-nitrophenyl phosphoric acid is fit to use with the alkaline phosphatase conjugate; For the peroxidase conjugated thing, use 1 usually, 2-phenylenediamine or Tolylamine.Also can use fluorogenic substrate, it generates fluorescence-causing substance rather than chromogenic substrate mentioned above.The solution that will contain suitable substrate then adds in the ternary complex.Substrate be connected to the enzyme reaction of second antibody, produce visible signal qualitatively, this signal can be further by spectrophotometer normally quantitatively to estimate the protein content that exists in the serum sample.

Alternatively, can with fluorescent chemicals (for example fluorescein and rhodamine) under the condition of the binding ability that does not change antibody chemical coupling to antibody.After the rayed of utilizing specific wavelength activated, the antibody of fluorochrome mark absorbed luminous energy, and inducing molecule enters excited state, and launches feature long wavelength's light subsequently.This emission light shows as observable characteristic color under opticmicroscope.Immunofluorescence and EIA technology in this area all be very proven technique and for present method institute preferred especially.Yet the present invention also can use other reporter molecules, for example radio isotope, chemoluminescence or bioluminescent molecules.To those skilled in the art, how changing method is conspicuous to adapt to required purposes.

Also can translate the mensuration of state according to several other methods.For example, by making up the protein monitoring (i.e. " proteomics " that microarray can carry out whole genome, Goffeau etc., above quoted passage), wherein the binding site of this microarray comprises the specific antibody of a large amount of kinds of protein tools immobilized, the pair cell genome encoding, preferred monoclonal antibody.Preferably, there is antibody, or exists at least and test or those relevant proteinic antibody of checking purpose bio-networks model at quite most coded protein.The preparation monoclonal antibody method be known (referring to, for example Harlow and Lane, " Antibodies:A Laboratory Manual ", Cold SpringHarbor, NY (1988), for all purposes, it is reference that integral body is incorporated this piece document into).In a preferred embodiment, produce the pulsating monoclonal antibody of anti-synthetic peptide, described synthetic peptide fragment is based on the cellular genome sequences Design.When using this antibody array, make protein contact array, and detect their combination with assay method known in the art from cell.

Alternately, can pass through two-dimensional gel electrophoresis system isolated protein.Two-dimensional gel electrophoresis is well known in the art, generally include along the unidimensional isoelectrofocusing, subsequently along second the dimension the SDS-PAGE electrophoresis (referring to, Hames etc. for example, " Gel Electrophoresis of Proteins:APractical Approach ", IRL Press, NY (1990); Shevchenko etc., Proc.Nat ' lAcad.Sci.USA, 93 volumes, 1440-1445 page or leaf (1996); Sagliocco etc., Yeast, 12 volumes, 1519-1533 page or leaf (1996); Lander, Science, 274 volumes, 536-539 page or leaf (1996))).Can comprise mass-spectrometric technique by many technology, western trace and the immunoblotting assay method of utilizing polyclone and monoclonal antibody and the inner and resulting electrophoretogram of the terminal micrometering preface methods analyst of N-.Utilize these technology, can identify under given physiological condition (comprise (for example, in the yeast) in the cell that is exposed to medicine, or by for example disappearance or cross and express in the cell that specific gene modifies) all proteins that produces quite most of.

Embodiment based on the others of biological condition

Although there are some technical difficulties that do not run at present in the cellular component beyond the monitoring mRNA abundance when monitoring mRNA, but it will be apparent for a person skilled in the art that, when using the inventive method, can measure and cell function feature proteins associated matter activity, and embodiment of the present invention can be measured based on this.The method of any functional, biochemical or physics that can be by being suitable for given activity to be characterized is carried out determination of activity.When activity relates to chemical conversion, cell protein can be contacted with natural substrate, and measure conversion rate.When activity relates to contact in the polymer unit, for example activatory DNA in conjunction with the combining of mixture and DNA the time, can measure the amount of related protein or the secondary result of this contact, as the mRNA that transcribes amount.And, when only knowing functionally active, for example during the functionally active in the cell cycle control, realization that can overview function.Yet no matter how knownly and measurement be, the variation of protein active can form the reply data that aforesaid method of the present invention is analyzed.

In alternative non-limiting embodiments, reply data can be made up of the mixing aspect of cell biological state.Can make up reply data from the variation of for example some mRNA abundance, the variation of some protein abundance and the variation of some protein active.

Computer realization

In preferred embodiments, in order to be provided for forming and the powerful of detection of biological system model and instrument easily, on the computer system or carry out the calculation procedure of preceding method on the computer system of one or more networkings.Computer system can be the single hardware platform that comprises inner member and be connected with outer member.The inner member of this computer system comprises and the interconnected processor elements of primary storage.For example computer system can have 200Mhz or more treater and the 32MB or the bigger primary storage based on IntelPentium of scale clock frequency.

Outer member comprises large capacity data memory.This mass storage can be one or more hard disks (its usually and treater be in the same place with memory device).Usually, this hard disk provides the storage of 1GB at least.Other outer member comprises user interface apparatus, and this can be indicating meter and keyboard and pointing device (this can be " mouse ") or other graphic input device.Usually, this computer system also with the communication network of other local computer system, remote computer system or wide area, connect as the internet.This network connects this computer system of permission and other computer system is shared data and Processing tasks.

Be downloaded to storer at the several softwares of this system's run duration, they in this area be standard and be specific to the present invention.The method according to this invention, these component softwares jointly make computer system work.These component softwares are stored on the mass storage usually.Alternatively, can be on removable medium such as floppy disk or CD-ROM (not shown) store software components.These component softwares are represented operating system, and this operating system is in charge of department of computer science's its network interworking of unifying.This operating system can be, Microsoft Windows family for example, and as Windows95, Windows98 or Windows NT, or Unix operating system are as Sun Solaris.Software comprises that the common-use words that are suitable for being present in this system function of making peace realizes specific method of the present invention with helper.The language of analytical procedure of the present invention of can being used to programme comprises C, C ⁺⁺Or JAVA not too preferably.Most preferably, with mathematical software bag programming the inventive method, the high-level explanation of this mathematical software bag admissible mark input equation formula and processing, it comprises the algorithm that will use, so the user does not need sequencing ground one equation of programming or algorithm.This software package comprises for example MATLAB of Mathworks (Natick MA) ^TM, Wolfram Research (Champaign, MATHEMATICATM IL) and Mathsoft (Cambridge, MATHCAD MA) ^TM

In preferred embodiments, in fact the analysis software assembly comprises interactional independent component software.The analysis software representative comprises the database that system moves all essential data.This data generally comprise, but are not result, genomic data, experimental procedure and expense and other information of test before must being limited to, and these it will be apparent to those skilled in the art that.Analysis software comprises data reduction and calculating composition, and this calculating composition comprises the program of one or more execution analytical procedure of the present invention.Analysis software also comprises user interface (UI), and this interface makes computer system user can control and the input test network model, and alternatively, experimental data.User interface can comprise the drag-and-drop interface that is used for the illustrative system hypothesis.User interface also can comprise and from the large vol memory module (for example being used for, hard-drive), from removable medium (for example floppy disk or CD-ROM) or by network (for example, local area network or Wide Area Communication network such as internet) from being written into the method for testing data with the various computing machine system of native system communication.

The present invention also provides to make up and has comprised at least one mark of the present invention, for example method of the database of mRNA or protein.For example, polynucleotide or aminoacid sequence can be stored in the digital storage media so that can the process of compilation system can identify the gene of breast cancer cell with standardized performance.This data handling system is useful to analyzing two intercellular genetic expressions, wherein at first selects one and is suspect to be and has tumour phenotype or genotypic cell, separates polynucleotide then from this cell.Isolating polynucleotide check order.With the sequence that exists in homology search technology comparative sample sequence and the database.Greater than 90%, more preferably, greater than 95%, more preferably, the sequence identity more than or equal to 97% shows from the front: these polynucleotide are separated from breast cancer cell as defined above between test sequence and polynucleotide sequence of the present invention.

The alternative computer system and method for realizing analytical procedure of the present invention it will be apparent to those skilled in the art, and is included in the claims.Particularly, claims are intended to comprise the alternative program structure that realizes the inventive method that these program structures it will be apparent to those skilled in the art that.

Modify abundance or the active method of mRNA

In the various embodiments of the present invention, produce useful clinically effect by changing or modifying abundance or the activity of expressing mRNA.At present, modify RNA abundance and active method and comprise 4 classes: ribozyme, antisense thing, double-stranded RNA and RNA fit (aptamer) (Good etc., Gene Therapy, 4 volumes, 45-54 page or leaf (1997)).The cell controllability used or be exposed to these entities allow the controllably abundance of RNA interfering, comprise mRNA abundance and activity, described activity comprises the translation of mRNA to active or detectable gene expression product (being protein).

Ribozyme

Ribozyme is the RNA molecule with mode special cutting other single stranded RNA similar to the DNA restriction endonuclease.Ribozyme has ability (Cech, Science, 236 volumes, the 1532-1539 page or leaf (1987) of catalysis RNA cleavage reaction; October 4 nineteen ninety disclosed PCT international publication WO90/11364; Sarver etc., Science, 247 volumes, 1222-1225 page or leaf (1990)).As Cech, Amer.Med.Assn., 260 volumes, 3030 pages (1988) are described, by modifying the nucleotide sequence of coding RNA, can synthesize ribozyme with specific nucleotide sequence in the identification molecule and with its cutting.Therefore, the mRNA that only has a particular sequence is cut and inactivation.

The ribozyme of two kinds of base types comprise " tup " type (as, Rossie etc., Pharmac.Ther., 50 volumes are described in the 245-254 page or leaf (1991)) and " hair clip " type ribozyme (as Hampel etc., Nucl.Acids Res., 18 volumes, 299-304 page or leaf (1999) and U.S. Patent number 5,254 are described in 678).Can design hair clip and tup RNA ribozyme with the specific said target mrna of special cutting.Establish design and had the rule of the short rna molecule of ribozyme activity, this RNA molecular energy cuts other RNA molecule in height sequence-specific mode, and can target the RNA of all kinds (Haseloff etc., Nature in fact, 334 volumes, 585-591 page or leaf (1988); Koizumi etc., FEBS Lett., 228 volumes, 228-230 page or leaf (1988); Koizumi etc., FEBS Lett., 239 volumes, 285-288 page or leaf (1988)).

The ribozyme method relate to make cellular exposure in this little RNA ribozyme molecule with the expression in the inducing cell, wait (Grassi and Marini, Annals of Medicine, 28 the volume, 499-510 page or leaf (1996); Gibson, Cancer and Metastasis Reviews, 15 volumes, 287-299 page or leaf (1996)).Can be by target corresponding to the tup of the mRNA of at least a open gene and hair clip ribozyme in intracellular expression to suppress the protein of this genes encoding.

Can directly send ribozyme with the RNA oligonucleotide form that comprises ribozyme sequence, perhaps introduce ribozyme to cell with the expression vector form of coding expectation ribozyme rna to cell.Ribozyme can be in vivo with enough effectively the quantity of catalyze cleavage mRNA express routinely, therefore modify mRNA abundance in the cell (referring to, Cotton etc., " RNA destroys in the body of nucleic acid mediation ", The EMBO J., 8 volumes, 3861-3866 page or leaf (1989)).Especially, can be with according to former Rule Design, and for example be connected in the Restriction Enzyme site of the anticodon stem of gene of coding tRNA and ring, transform it by this area ordinary method then and enter the purpose cell and in cell, express by standard phosphoramidite chemical method synthetic ribozyme DNA sequences encoding.Preferably, also introduce inducible promoter (for example, glucocorticosteroid or tsiklomitsin response element), can selectively control ribozyme like this and express to this construct.For saturated application, can utilize to have the highly active promotor of composing type.Because (that is) small size and the high transcription rate in the different sorts tissue and generally expression, the gene of coding tRNA is so be useful in this application for the tDNA gene.

Therefore, can design routinely ribozyme with the cutting in fact any mRNA sequence, and can with the coding this ribozyme sequence DNA routinely transformant with the ribozyme of controlled expression catalytically effective amount.Therefore, can modify or interference cell in the abundance of in fact any RNA thing class.

Can use and modify ribozyme sequence with regard to the essentially identical mode of the described mode of antisense nucleotide, for example ribozyme sequence can contain the base portion of modification.

Antisense molecule

In another embodiment, can controllably suppress the activity of target RNA (preferred mRNA) kind, particularly its translation speed by the controlled application of antisense nucleic acid.High level is used and is caused saturated inhibition.Here used " antisense " nucleic acid refer to can by means of with certain sequence of coding and/or non-coding region complementary and with the sequence specific (for example non-polyA) of the target RNA nucleic acid of (for example its translation initiation district) hybridization partly.Antisense nucleic acid of the present invention can be two strands or single stranded oligonucleotide, can be RNA or DNA or its modifier or derivative, and can directly use for cell with controllable manner, perhaps can in cell, produce by the controlled amounts of transcribing-translating of external source calling sequence with enough interference target RNA.

Preferably, antisense nucleic acid has 6 Nucleotide at least, preferably oligonucleotide (scope from 6 to about 200 oligonucleotide).In particular aspects, oligonucleotide has at least 10 Nucleotide, at least 15 Nucleotide, at least 100 Nucleotide, or at least 200 Nucleotide.Oligonucleotide can be DNA or RNA or their chimeric mixture or derivative or modified forms, and can be strand or two strands.Can be at base portion, sugar moieties or phosphoric acid backbone modification oligonucleotide.Oligonucleotide can comprise other additional group, as peptide or help cross-cell membrane transhipment the factor (referring to, Letsinger etc. for example, Proc.Natl.Acad.Sci.USA, 86 volumes, 6553-6556 page or leaf (1989); Lemaitre etc., Proc.Natl.Acad.Sci.USA, 84 volumes, 648-652 page or leaf (1987); On December 15th, 1998 disclosed PCT publication number .WO 88/09810), the cutting agent that triggers of hybridization (referring to, Krol etc. for example, BioTechniques, 6 volumes, 958-976 page or leaf (1988)) or intercalator (referring to, Zon for example, Pharm.Res., 5 volumes, 539-549 page or leaf (1988)).

The present invention preferred aspect, the antisense oligonucleotide that is preferably single stranded DNA is provided.Can on any position of oligonucleotide structure, modify it with component well known in the art.

Typical antisense method comprises the preparation with the mRNA complementary oligonucleotide (DNA or RNA) of genes encoding.Antisense oligonucleotide will be hybridized with the mRNA of this genes encoding, and stop translation.The ability of antisense base sequences and expectation gene recombination will depend on the complementary degree and the length of antisense base sequences.Usually, increase with hybrid nucleic acid length, it can contain and manyly still forms stable duplex or triple helix with the RNA mismatched bases.By utilizing ordinary method to measure the hybridization complex fusing point, those skilled in the art can determine the tolerance degree of mispairing.

Preferably, design and mRNA 5 ' end, for example, non-translated sequence up to and comprise mRNA initiation site, the i.e. antisense nucleotide of the regional complementarity of AUG.Yet, as Wagner, Nature, 372 volumes, 333 pages (1994) are described, also are proved with mRNA 3 ' non-translated sequence complementary oligonucleotide sequence and can suppress the mRNA translation effectively.Although can design and mRNA coding region complementary antisense oligonucleotide, this oligonucleotide is not too effective translational inhibitor.

Antisense oligonucleotide can comprise the base portion of at least one modification, the base portion of described modification is selected from and includes but not limited to: 5 FU 5 fluorouracil, 5-bromouracil, the 5-chlorouracil, 5-iodouracil, xanthoglobulin, xanthine, the 4-acetylcytosine, 5-(carboxyl hydroxymethyl) uridylic, 5-carboxyl methyl aminomethyl-2-thiouracil, 5-carboxyl methyl aminomethyl uridylic, dihydrouracil, β-D-semi-lactosi queosine, Trophicardyl, the N6-isopentennyladenine, the 1-methyl guanine, 1-methyl inosine, 2, the 2-dimethylguanine, the 2-methyladenine, 2-methyl guanine, 3-methylcystein, 5-methylcytosine, the N6-VITAMIN B4,7-methyl guanine, 5-methylamino 6-Methyl Uracil, 5-methoxyl group amino methyl-2-thiouracil, β-D-seminose queosine, 5 '-methoxyl group carboxyl 6-Methyl Uracil, the 5-methoxyuracil, 2-methylthio group-N6-isopentennyladenine, uridylic-5-fluoroacetic acid (v), wybutoxosine, pseudouridine, queosine, 2-sulfo-cytosine(Cyt), 5-methyl-2-thiouracil, 2-thiouracil, the 4-thiouracil, methyl uracil, uridylic-5-oxy acetic acid methyl ester, uridylic-the 5-fluoroacetic acid is (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2 carboxyl propyl group) uridylic, (acp3) w and 2,6-diaminopurine.

In another embodiment, oligonucleotide comprises the sugar moieties of at least one modification, and the sugar moieties of described modification is selected from but is not limited to: pectinose, 2-fluoro pectinose, xylulose and hexose.

In a further embodiment, oligonucleotide comprises the phosphoric acid skeleton of at least one modification, the phosphoric acid skeleton of described modification is selected from: thiophosphatephosphorothioate, phosphorodithioate, the sulfo-amino phosphoric acid ester, phosphoramidate, phosphorodiamidite, methylphosphonate, alkyl phosphotriester and formacetal or its analogue.

In a further embodiment, oligonucleotide is a 2-a-end group isomery oligonucleotide.A-end group isomery oligonucleotide and complementary RNA form special double-stranded heterozygote, and wherein the B-unit with common is opposite, moving towards of chain (Gautier etc., Nucl.Acids Res., 15 volumes, 6625-6641 page or leaf (1987)) parallel to each other.

Oligonucleotide can with other molecule, for example the cutting agent that triggers of the linking agent, transport agents, the hybridization that trigger of peptide, hybridization etc. is puted together.

Antisense nucleic acid of the present invention comprises at least a portion complementary sequence with target RNA thing.Yet although preferred complementary fully, this is optional.Mean as the sequence of " with the complementation of RNA at least a portion " mentioned here and enough can form the sequence that stablize double-helical complementarity with RNA hybridization; Under the situation of double-stranded antisense nucleic acid, can detect the strand of duplex DNA, maybe can measure the formation of triple helix.The hybridization ability will depend on complementary degree and antisense nucleic acid length.Usually, hybrid nucleic acid is long more, and it can comprise many more and target RNA mismatched bases and still form and stablize duplex (or under the possible situation, triple helix).By utilizing standard program to measure the fusing point of hybridization complex, those skilled in the art can determine the tolerance degree of mispairing.Can determine effectively to suppress the antisense nucleic acid amount of target RNA translation by the standard test technology.

Can be by standard method known in the art, for example by using (as from Biosearch, Applied Biosystems etc. is purchased) synthetic oligonucleotide of the present invention of automatic dna synthesizer.For example, can be by Stein etc., Nucl.Acids Res., 16 the volume, the method for 3209 pages (1988) is synthesized the thiophosphatephosphorothioate oligonucleotide, can prepare by the application of controlled porose glass, polymer carrier the methylphosphonate oligonucleotide (referring to, Sarin etc., Proc.Natl.Acad.Sci.USA, 85 volumes, 7448-7451 page or leaf (1988)) etc.).In another embodiment, oligonucleotide is 2 '-O-methyl ribonucleotides (Inoue etc., Nucl.Acids Res., 15 volumes, 6131-6148 page or leaf (1987)), or chimeric RNA-DNA analogue (Inoue etc., FEBS Lett., 215 volumes, 327-330 page or leaf (1987)).

Can use the synthetic antisense oligonucleotide to cell with controlled or saturation mode then.For example, can be with controlled horizontal positioned antisense oligonucleotide in the cell growing environment, cell can absorb described antisense oligonucleotide from this environment.Can help the picked-up of antisense oligonucleotide by the application of approach well known.

When antisense oligonucleotide was introduced into host cell, it was hybridized with for example, by suppressing the intracellular expression that suppresses coded protein of transcribing and/or translate with cell mRNA and/or genomic dna corresponding to gene specifically.

Can for example send the isolated nucleic acid molecule that comprises antisense base sequences with the form of expression vector, when when transit cell is recorded, it will produce at least one the differentiated part complementary RNA with the mRNA of genes encoding.Alternately, the isolated nucleic acid molecule that comprises antisense base sequences is the oligonucleotide probe of prepared ex vivo, and when its is introduced cell, hybridizes the expression by inhibitation that will cause coded protein by it and the mRNA and/or the genome sequence of this gene.

Preferably, oligonucleotide contains key between artificial nucleotide, and it makes antisense molecule have the resistance to exonuclease and endonuclease, and is therefore stable in cell.For example, as in U.S. Patent number 5,176,996; 5,264,564; With 5,256,775 is described, and the nucleic acid molecule of modification is phosphoramidate, thiophosphatephosphorothioate and the methyl-phosphorous acid ester analogs of DNA as the example of antisense base sequences.For example, at Van der Krol., BioTechniques, 6 volumes, 958-976 page or leaf (1988); With Stein etc., Cancer Res., 48 volumes have been described the general method that preparation is used for the oligomer of antisense therapy in the 2659-2668 page or leaf (1988).

The antisense molecule of cell inner expression

As mentioned above, can in the cell paste of expressing said gene, send antisense nucleotide by various technology, described technology comprises that for example the direct injection antisense nucleotide enters the mammary tissue site, bag carries an antisense nucleotide in liposome, uses the modified antisense Nucleotide of target mammary gland cell in conjunction with the acceptor of cell surface expression or antigenic peptide or antibody by connecting antisense nucleotide and specificity.

Yet the IC that utilizes above-mentioned delivering method to obtain the endogenous mRNA translation of enough inhibition may be difficult.Therefore, in alternative embodiment, the nucleic acid that will comprise antisense base sequences places transcribing under the control to form expression construct of promotor (that is, initial specific gene is transcribed required dna sequence dna).In cell, controllably express antisense nucleic acid of the present invention by transcribing from exogenous array.If control high-caliber expression, will occur saturated interference so and modify the result.For example, can introduce carrier in the body, this like cell will absorb this carrier, and carrier or its part are transcribed and produced antisense nucleic acid of the present invention (RNA) in cell.This carrier will contain the sequence of encoding antisense nucleic acid.As long as carrier can be transcribed the sense-rna that produces expectation, this carrier can keep free type or integrate on karyomit(e).Can make up this carrier by this area standard recombinant dna technological method.Carrier can be a plasmid, and viral or known in the art other can be used for the carrier that mammalian cell duplicates and expresses.Can be by the expression of the sequence of encoding antisense RNA in any promotor starting purpose cell known in the art.This promotor can be induction type or composing type.Most preferably, promotor can be controlled or induces by using of external source part, to realize the controlled expression of antisense oligonucleotide.This controllable initiating comprises the Tet promotor.Other promotor that can be used for mammalian cell include but not limited to SV40 early promoter zone (referring to, Bernoist and Chambon, Nature, 290 volumes, 304-310 page or leaf (1981)), Rous sarcoma virus 3 ' the long terminal repetition promotor that contains (Yamamoto etc., Cell, 22 volumes, 787-797 page or leaf (1980)), herpesvirus thymine deoxyriboside kinase promotor (Wagner etc., Proc.Natl.Acad.Sci.USA, 78 volumes, 1441-1445 page or leaf (1981)), metallothionein gene is regulated sequence (Brinster etc., Nature, 296 volumes, 39-42 page or leaf (1982)) etc.

Therefore, can the conventional design antisense nucleic acid with any mRNA sequence of target in fact, can be with the nucleic acid transformant routinely of this antisense sequences of coding, or with the nucleic acid of cellular exposure in this antisense sequences of coding, can express so effectively and the antisense nucleic acid of controlled or saturation capacity.Therefore, can modify or interference cell in the translation of in fact any RNA kind.

Double-stranded RNA

Also can utilize double-stranded RNA, promptly have justice-sense-rna to disturb at least a open expression of gene corresponding at least a open gene.Verified various organisms, as can disturbing the function and the expression of native gene in the Caenorhabditis elegans (C.elegans) by double-stranded RNA, referring to as Fire etc., Nature, 391 volumes are described in the 806-811 page or leaf (1998).

RNA is fit

At last, in an embodiment again, can in cell, introduce or expressed rna fit.RNA is fit to be proteinic special RNA part, and as the RNA (Good etc., GeneTherapy, 4 volumes, 45-54 page or leaf (1997)) at Tat and Rev, it can suppress their translation specifically.

Modify the abundance or the active method of marking protein

The method of modifying protein abundance especially comprises method that changes protein degradation speed and the method (thereby antibody binding proteins matter influences the activity or the abundance of natural target protein pledge) of utilizing antibody.Directly the active method of modifying protein especially comprises the application of antibody, dominance negative sudden change, specific drugs or chemical part.

Increase (or reduction) a kind of proteinic degradation rate and can reduce (or increasing) proteinic abundance of this kind.Temperature by replying rising and/or the method that is exposed to specific medicine and increases the target protein degradation rate are known in the art, can utilize this method in the present invention.For example, a kind of method has been utilized thermal induction or drug-induced N-end degron, it is a N-end protein matter fragment, expose the degraded signal in this fragment of comparatively high temps (for example 37 ℃) and promote the fast protein degraded, this fragment of lesser temps (for example 23 ℃) be hidden with stop quick degraded (referring to, Dohmen, Science, 263 volumes, 1273-1276 page or leaf (1994)).The example of this degron is Arg-DHFR ^TsThe varient of-Muridae Tetrahydrofolate dehydrogenase is wherein at the Pro of terminal Arg displacement Val of N-and Leu displacement position 66.According to this method, can be for example, by standard gene targeted approach (Lodish etc. known in the art, " Molecular Biology of the cell ", W.H.Freeman and Co., NY (1995), the 8th chapter particularly), with encoding fusion protein matter Ub-Arg-DHFR ^TsThe gene of the gene substitution target protein P of-P (" Ub " represents ubiquitin, and " P " represents target protein).Thereby the ubiquitin of N-end is exposed the terminal degron of N-by fly-cutting after translation.When lesser temps, do not expose Arg-DHFR ^TsThe ubiquitination of fused protein does not appear in inner Methionin, and degraded is slow, and active target protein level height.In comparatively high temps (under the non-existent situation of methotrexate), expose Arg-DHFR ^TsInner Methionin, ubiquitination appears in fused protein, and degraded is quick, and active target protein level is low.

Because the thermal activation of degraded can controllably be blocked by exposing methotrexate, so this technology also allows the controlled modification of degradation rate.This method is applicable to other induction factor, produces the terminal degron of other N-that replys as medicine and temperature variation.And the expression that it will be appreciated by those skilled in the art that the antibody that can utilize combination and suppress target protein is as the negative strategy of another dominance.

With small-molecule drug or ligand modified expressed protein activity

In addition, can modify or disturb the activity of some target protein with controlled or saturation mode by being exposed to external source medicine or part.Because the inventive method usually is applied to test or proves the availability of various medicines to the treatment cancer, so drug exposure is the important method of modification/interference cell component-mRNA and expressed protein.In preferred embodiments, disturb the input cellular component, and measure systems response by gene expression technique (as following hybridization) to genetic transcription thing array by drug exposure or genetic manipulation (as genetically deficient or rejecting).

Under a kind of preferred situation, only a kind of target protein interacts in known drug and the cell, and changes the only activity of this a kind of target protein, promptly or increase activity or reduce this activity.Therefore, cell contacts with the gradient of this medicine of variable quantity and will cause this target protein is wherein disturbed as the gradient of the network model of input thing.Saturated exposure causes saturated modification/interference.For example, cyclosporin A is the very special proteinic conditioning agent of calcineurin, and it works by cyclophilin is compound.So the cyclosporin A that can utilize a series of titres produces the calcineurin protein restraining effect of any desired intensity.Alternately, the saturated cyclosporin A that is exposed to will farthest suppress calcineurin protein.

With antibody and antagonist modifying protein activity

Term " antagonist " refers to can suppress its active molecule when with the protein bound of genes encoding.Antagonist includes but not limited to peptide, protein, carbohydrate and small molecules.

In useful especially embodiment, antagonist is the antibody special to the cell surface proteins of at least a genetic expression.Antibody as therapeutical agent comprises above-mentioned antibody.The effector that antibody can be used as treatment separately works, or it can enlist other cell in fact to realize cell killing.Antibody also can with reagent, as chemotherapeutics, radionuclide, ricin A chain, Toxins,exo-, cholera, Toxins, pertussiss etc. are puted together, and as directed medicament.Alternately, effector can be to have directly or indirectly and the lymphocyte of the interactional surface molecular of tumour target.Various effector cells comprise cytotoxic T cell and NK cell.

The example that is used in the antibody-therapeutical agent conjugate in the treatment includes, but are not limited to:

1) with radionuclide as ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶HO ', ¹⁷⁷LU, ¹⁸⁶Re and ¹⁸⁸Re link coupled antibody, and can referring to as, Goldenberg etc., Cancer Res., 41 the volume, 4354-4360 page or leaf (1981); Carrasquillo etc., Cancer Treat.Rep., 68 volumes, 317-328 page or leaf (1984); Zalcberg etc., J.Natl.Cancer Inst., 72 volumes, 697-704 page or leaf (1984); Jones etc., Int.J.Cancer, 35 volumes, 715-720 page or leaf (1985); Lange etc., Surgery, 98 volumes, 143-150 page or leaf (1985); Kaltovich etc., J.Nucl.Med., 27 volumes, 897 pages (1986); Order etc., Int.J.Radiother.Oncol.Biol.Phys., 8 volumes, 259-261 page or leaf (1982); Courtenay-Luck etc., Lancet, 1 volume, 1441-1443 page or leaf (1984); With Ettinger etc., Cancer Treat.Rep., 66 volumes are described in the 289-297 page or leaf (1982);

2) with medicine or biological response modifier, as methotrexate, Zorubicin and lymphokine are as Interferon, rabbit link coupled antibody, referring to for example Chabner etc., " Cancer, Principles and Practice of Oncology ", J.B.Lippincott Co., Philadelphia, PA, 1 volume, 290-328 page or leaf (1985); Oldham etc., " Principles and Practice of Oncology ", Cancer, J.B.Lippincott Co., Philadelphia, PA, 2 volumes, 2223-2245 page or leaf (1985); Deguchi etc., Cancer Res., 46 volumes, 43751-43755 page or leaf (1986); Deguchi etc., Fed.Proc., 44 volumes, 1684 pages (1985); Embleton etc., Br.J.Cancer, 49 volumes, 559-565 page or leaf (1984); With Pimm etc., CancerImmunol.Immunother., 12 volumes are described in the 125-134 page or leaf (1982);

3) with toxin conjugated antibody, referring to for example Uhr etc., " Monoclonal Antibodiesand Cancer ", Academic Press, Inc., 85-98 page or leaf (1983); Vitetta etc., " Biotechnology and Bio.Frontiers ", P.H.Abelson edits, 73-85 page or leaf (1984); With Vitetta etc., Science, 219 volumes are described in the pp.644-650 page or leaf (1983);

4) exclusive-OR function antibody, for example with another antibody coupling or bonded antibody, like this mixture both in conjunction with cancer also in conjunction with the effector cell, for example killer cell such as T cell, referring to for example Perez etc., J.Exper.Med., 163 volumes, 166-178 page or leaf (1986); With Lau etc., Proc.Natl.Acad.Sci.USA, 82 volumes are described in the 8648-8652 page or leaf (1985); With

5) natural, promptly non-puting together or non-compound antibody, referring to for example, Herlyn etc., Proc.Natl.Acad.Sci.USA, 79 volumes, 4761-4765 page or leaf (1982); Schulz etc., Proc.Natl.Acad.Sci.USA, 80 volumes, 5407-5411 page or leaf (1983); Capone etc., Proc.Natl.Acad.Sci.USA, 80 volumes, 7328-7332 page or leaf (1983); Sears etc., Cancer Res., 45 volumes, 5910-5913 page or leaf (1985); Nepom etc., Proc.Natl.Acad.Sci.USA, 81 volumes, 2864-2867 page or leaf (1984); Koprowski etc., Proc.Nat.Acad.Sci.USA, 81 volumes, 216-219 page or leaf (1984); With Houghton etc., Proc.Natl.Acad.Sci.USA, 82 volumes are described in the 1242-1246 page or leaf (1985).

Coupling antibody or its fragment are well known in the art with the method for therapeutical agent as mentioned above, and have described these methods in the method that provides in the above referred-to references.

Antagonist is as the purposes of therapeutical agent

In a further embodiment, the antagonist as the agent of treatment breast cancer treatment can be a kind of proteinic inhibitor of open genes encoding.For example, can suppress the activity of membrane-bound serine protease hepsin, and this will be with the growth of the system toxicity blocking-up malignant galactophore cell of minimum by utilizing special serpin.This serpin is known in the art.For example, arotinin is that approved is used for reducing the blood loss of cardiopulmonary bypass surgery and the serpin of blood transfusion needs, it suppresses kallikrein and fibrinolysin, cause the multisystem that participates in inflammatory reaction inhibition (referring to, Ann.Thorac.Surg., 71 volumes, 2 phases, 745-754 page or leaf (2001)).

Maspin (breast Serpin) has the breast cancer tumour of inhibition novel serine protease inhibitor function, relevant with serpin family (referring to Acta.Oncol., 39 volumes, 8 phases, 931-934 page or leaf (2000)).

Zymoplasm and factor Xa (fXa) are the serine proteases of unique little, effective, non-covalent inhibitor of developing of selectivity, these inhibitor last minute plannings as the candidate of drug development (in this case, as anti-coagulant) (referring to, Med.Res.Rev., 19 volumes, 2 phases, 179-197 page or leaf (1999))..

Also can reduce the target protein activity by (neutralization) antibody.By the controlled or saturated exposure to this antibody is provided, can modify or interferencing protein abundance/activity with controlled or saturation mode.For example, can become with the wild-type of not assembling the wild-type form and relatively have the abundance that less or minimum active mixture reduces target protein wild-type activity form by assembling the target protein activity form at the antibody that is fit to epitope on the protein surface, reduce active thus indirectly.Alternately, antibody can pass through, and for example direct and avtive spot interacts or passes through the blocking-up substrate near avtive spot, directly reduces protein active.On the contrary, in some cases, the activity that (activation) antibody also can interact and be produced to increase with the avtive spot of protein and they.Under any circumstance, all can (by the method that will describe) produce (that will describe the is various types of) antibody of anti-specific protein kind and their effect is screened.For example can measure the effect of antibody, select to improve or reduce the concentration and/or the active suitable antibody of target protein kind.This mensuration comprises to cell introduces antibody (vide infra), and measures wildtype target protein concn or activity by standard method known in the art (as immunoassay).Can measure the clean activity of wild-type form by the measuring method that is suitable for the target protein known activity.

In cell, introduce antibody

Can antibody be introduced cell with many modes, comprise, for example microinjection antibody enter cell (referring to, Morgan etc., Immunology Today, 9 volumes, 84-86 page or leaf (1988)) or the hybridoma mRNA that transforms coding expectation antibody enter cell (referring to, Burke etc., Cell, 36 volumes, 847-858 page or leaf (1984)).In another technology, can artificial constructed recombinant antibodies and in extensively multiple non-lymphocyte type dystopy ground express with in conjunction with target protein and blocking-up target protein activity (Biocca etc., Trends in Cell Biology, 5 volumes, 248-252 page or leaf (1995)).Preferably, antibody expression is subjected to controllable initiating as Tet promotor or forms active promotor (being used to produce saturated interference) and control.The first step is that screening has suitably specific specific monoclonal antibody (vide infra) to target protein.Then, the sequence clone of the selected antibody variable region of coding is gone into range gene engineered antibody form, comprise, for example complete antibody, Fab fragment, Fv fragment, strand Fv fragment are (by the V of peptide linker connection _HAnd V _LThe zone) (" ScFv " fragment), diabodies (two combine have not homospecific ScFv fragment) or the like (Hayden etc., Current Opinion in Immunology, 9 volumes, 210-212 page or leaf (1997)).Various forms antibody expression by making cell inner expression for various known cells in the protein that merges of leader sequence, can make their orientations (for example enter cellular compartment, tenuigenin, nucleus and plastosome etc.) (Bradbury etc., Antibody Engineerinq, 2 volumes, Borrebaeck edits, 295-361 page or leaf, IRL Press (1995)).Especially, the ScFv form seems and is particularly suitable for the tenuigenin target.

Various useful antibody types

The antibody type includes but not limited to polyclone, mono-clonal, chimeric, single-chain antibody and Fab fragment and Fab expression library.The whole bag of tricks known in the art can be used to produce the polyclonal antibody of target protein.In order to produce antibody, can use the various host animals of target protein injecting immune, this host animal includes but not limited to rabbit, mouse and rat etc.Can be according to host species, utilize various adjuvants to increase immunne response, described adjuvant includes but not limited to human adjuvant such as the bacill calmette-guerin (BCG) and the corynebacterium parvum (Corynebacterium parvum) of Fu Shi (fully with incomplete) adjuvant, mineral coagulant such as aluminium hydroxide, surfactant such as lysolecithin, poly alcohol, polyanion, peptide, oil-emulsion, dinitrophenol(DNP) and potentially useful.

Monoclonal antibody

In order to prepare the monoclonal antibody of pointing to target protein, can utilize any technology that allows the continuous cell line cultivated to produce antibody molecule.This technology includes but not limited to Kohler and Milstein, Nature, 256 volumes, the hybridoma technology of 495-497 page or leaf (1975) original research exploitation, trioma technology and human B cell hybridoma technology (referring to, Kozbor etc., Immunology Today, 4 volumes, 72 pages (1983)) and produce the EBV hybridoma technology (Cole etc. of human monoclonal antibodies, " MonoclonalAntibodies and Cancer Therapy ", Alan R.Liss, Inc., 77-96 page or leaf (1985)).In other embodiment of the present invention, can utilize nearest technology in germ-free animal, to produce monoclonal antibody (PCT/US90/02545).According to the present invention, can utilize people's antibody, and can be by utilizing people's hybridoma (referring to, Cote etc., Proc.Natl.Acad.Sci.USA, 80 volumes, 2026-2030 page or leaf (1983)) or by with EBV virus vitro conversion human B cell (referring to, Cole etc., " MonoclonalAntibodies and Cancer Therapy ", Alan R.Liss, Inc., 77-96 page or leaf (1985)) acquisition people antibody.In fact, according to the present invention, can utilize " chimeric antibody " generating technique of exploitation, this technology will be by will implement (referring to Morrison etc. the gene splicing of the human antibody molecules of the gene of the special mouse antibodies molecule of target protein and the suitable biologic activity of tool together, Proc.Natl.Acad.Sci.USA, 81 volumes, 6851-6855 page or leaf (1984); Neuberger etc., Nature, 312 volumes, 604-608 page or leaf (1984); Takeda etc., Nature, 314 volumes, 452-454 page or leaf (1985)); This antibody within the scope of the present invention.

In addition, when monoclonal antibody when being favourable, alternatively can with display technique of bacteriophage from big antibody library select they (referring to, Marks etc., J.Biol.Chem., 267 volumes, 16007-16010 page or leaf (1992)).Utilize this technology, at fd filobactivirus surface expression nearly 10 ¹²The library of individual different antibodies, produce " single jar " the external antibody mediated immunity system can be used for the monoclonal antibody screening (referring to, Griffiths etc., EMBO J., 13 volumes, 3245-3260 page or leaf (1994)).Can be by technology known in the art, comprise allowing phage contact fixed target protein, screening and clone go into to express in the suitable carrier of expectation antibody formation, from this library, screen antibody in conjunction with the phage of target and with the sequence subclone of encoding antibody variable region.

According to the present invention, can adaptability revision U.S. Patent number 4,946,778 described single-chain antibody generating techniques to produce the single-chain antibody special to target protein.Other embodiment of the present invention has utilized Fab expression library constructing technology (referring to, Huse etc., Science, 246 volumes, 1275-1281 page or leaf (1989)) to allow to identify fast and easily target protein to be had the specific mono-clonal Fab fragment of expectation.

Can produce the antibody fragment of the target protein that comprises idiotype by technology known in the art.For example, this fragment F that includes but not limited to produce by the pepsin digested antibody molecule (ab ') ₂Fragment; Can be by reduction F (ab ') ₂Fab ' the fragment that segmental disulphide bridges produces; Can be by handling Fab fragment and the Fv fragment that antibody molecule produces with papoid and reductive agent.

In antibody produces, can be by technology known in the art, for example ELISA finishes the screening of expectation antibody.In order to screen the antibody special to target protein, hybridoma or the phage displaying antibody storehouse that can measure generation can be in conjunction with the antibody of target protein to seek.

Active other method of modifying protein

The negative sudden change of dominance is to destroy active native gene sudden change of target protein pledge or foreign gene mutant when expressing in cell.According to the structure and the activity of target protein, exist to select make up the general guide principle of the negative sudden change of dominance appropriate strategy, the negative sudden change of described dominance will destroy target activity (referring to, Hershkowitz, Nature, 329 roll up 219-222 page or leaf (1987)).Under the situation of reactive monomer form, the overexpression of inactivation form can cause is enough to reduce significantly the clean active competition to natural substrate or part of target protein.By, for example connection has the active promotor of increasing, and preferably controlled or inducible promoter, or composition type expression promoter and mutant gene can be realized this overexpression.Alternately, can carry out change, be connected thereby cause with target ligands is in fact irreversible to the avtive spot residue.This can be by the serine residue in careful substitute activity site, with some Tyrosylprotein kinase realize (referring to, Perlmutter etc., Current Opinion in Immunology, 8 volumes, 285-290 page or leaf (1996)).

Under the situation of many subunits of activity form, there are several strategies can instruct the selection of dominance being born mutant.Can reduce the activity of many subunits with controlled or saturation mode by the gene of expressing the encoding exogenous protein fragments, described exogenous protein fragment can and stop polymer to form in conjunction with many subunits relational structure territory.Alternately, the unitary controlled or saturated overexpression of particular type inactivating proteins can be strapped in the wild-type activity unit in the inactivation polymer, therefore reduce the polymer activity (referring to, Nocka etc., EMBO J., 9 volumes, 1805-1813 page or leaf (1990)).For example, under the situation of dimer dna binding protein dna, can from the DNA combining unit, lack the DNA binding domains, perhaps from activation unit, lack activation domain.And, in this case, can express such DNA binding domains unit, this structural domain unit does not cause and activation unit bonded structural domain.Therefore, take the DNA binding site, and without any activating expression.

Unit for particular type during activity normally will carry out the situation of conformational change, the mixture that the expression of rigid element can inactivation obtains thus.Again for example, participate in cell mechanism, normally combine and form by many subunits that belong to several types as the protein of cell movement, mitotic division process, cell construction etc.These structures are usually extremely sensitive to the destruction that several monomeric units with textural defect are included.This sudden change monomer will destroy the related protein activity, can express with controlled or saturation mode in cell.

Except the negative sudden change of dominance, can find the sudden change target protein responsive to temperature (or other exogenous factor) by mutagenesis well known in the art and screening method.

Form of therapy

Under situation with the antisense nucleotide treatment, this method comprises the isolated nucleic acid molecule for the treatment of significant quantity, this isolated nucleic acid molecule comprises and derives from table 1,2, the antisense base sequences of at least a gene of identifying in 3 or 4, wherein this antisense nucleotide ability of described at least a gene transcription/translation that changes.Term " isolating " nucleic acid molecule means the nucleic acid molecule that (when nucleic acid molecule is natural, then being natural surroundings for example) shifts out from its primal environment.For example, naturally occurring nucleic acid molecule is not isolating, but for isolated identical nucleic acid molecule in the some or all of coexisting substances from natural system, even it will be introduced into this natural system subsequently again, it also is isolating.This nucleic acid molecule can be the part of carrier or the part of composition, and still is isolating, because this carrier or composition are not the parts of the natural surroundings of nucleic acid molecule.

Aspect ribozyme or double stranded rna molecule treatment, this method comprises the nucleotide sequence or the double stranded rna molecule of the encoding ribozyme for the treatment of significant quantity, wherein nucleotide sequence/the double stranded rna molecule of the encoding ribozyme ability of described at least a gene transcription/translation that changes.

With under the situation of antagonist for treating, this method comprises the antagonist that gives the patient treatment significant quantity, and this antagonist can suppress or activate table 1, the protein of at least a coded by said gene of evaluation in 2,3 or 4.

" the treatment significant quantity " of nucleotide sequence, double-stranded RNA or antagonist that comprises isolated nucleic acid molecule, the encoding ribozyme of antisense nucleotide is meant that one of these therapeutical agents are enough to treat the amount of mammary cancer (for example limit growth of breast cancers or delay or stop metastases).The treatment significant quantity fixes in those skilled in the art's the limit of power really.For any treatment, can be in the cell culture of for example neoplastic cell or at animal model, normally mouse, rabbit, the treatment effective dose is estimated in preliminary survey in dog or the pig.Also can determine the suitable concentration range and the approach of administration with animal model.Useful dosage and approach in the time of can utilizing these information to determine human administration then.

Can pass through the standard pharmacological method, in cell culture or laboratory animal, measure treatment and render a service and toxicity, for example ED ₅₀(the effective dosage of treatment in 50% colony) and LD ₅₀(causing colony's 50% lethal dosage).Dosage rate between toxic action and therapeutic action is a therapeutic index, and it can be expressed as ratio LD ₅₀/ ED ₅₀Preferred big treatment exponential antisense nucleotide, ribozyme, double-stranded RNA and the antagonist of showing.When formulating the human dosage range, can utilize from the data of cell culture assays and zooscopy acquisition.The dosage that contains in this composition is preferably in following circulation composition scope, and this circulation composition scope comprises and has little or no toxic ED ₅₀According to used dosage form, patient's susceptibility and route of administration, dosage can change in this scope.

According to the relevant factor of patient of needs treatments, the doctor can determine definite dosage.Can adjust dosage and dosage regimen with active part that enough levels are provided or keep desired effects.The factor that may consider comprises the seriousness of patient disease state, total health, patient's age, body weight and sex, diet, administration time and frequency, medication combined, reaction sensibility and to the tolerance of treatment/reply.

According to route of administration, normal dosage can from 0.1 to 100,000 microgram, up to the total dose of about 1g.Provide guidance in the literature, and the practitioner of this area generally can obtain this guidance about given dose and delivering method.Those skilled in the art will use different preparations with antagonist to Nucleotide.

Use for treatment, the nucleotide sequence of preferred antisense nucleotide, encoding ribozyme, double-stranded RNA (no matter wrap to carry in liposome and also be included in the virus vector) and antibody are as the pharmaceutical composition administration, and this pharmaceutical composition will contain one or more pharmaceutically acceptable carriers and described therapeutical agent.Can give separately said composition or with at least a other medicament, as the stable compound Combined Preparation; Can include but not limited to salt solution at any aseptic, biocompatible pharmaceutical carrier, buffer saline is used said composition in glucose and the water.Can give the patient said composition separately, perhaps also give other medicament of patient, medicine or hormone.

Can pass through number of ways, include but not limited to oral, intravenously, intramuscular, intraarticular, intra-arterial, in the marrow, in the sheath, in the ventricle, transdermal, subcutaneous, intraperitoneal, in the nose, enteron aisle, part, hypogloeeis or the rectal pharmaceutical composition that gives.Except active ingredient, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carrier, include to be beneficial to active compound is processed as the pharmaceutically vehicle and the auxiliary agent of available preparation.Can be at " Remington ' sPharmaceutical Sciences " of recent release, Maack Publishing Co., Easton finds the more details about preparation and medicine-feeding technology among the PA..

Utilize the pharmaceutically acceptable carrier that is suitable for oral administration well known in the art, can prepare pharmaceutical composition for oral administration fully.This carrier makes pharmaceutical composition can be mixed with the tablet that can be digested by the patient, pill, drageeing, capsule, liquid, gel, syrup, slurries, suspension etc.

Can obtain pharmaceutical preparation for oral use in the following way: mixed active compound and solid excipient, alternatively, grind the mixture that obtains thus, and the processing granular mixture (if expectation, after adding suitable auxiliary agent), obtain tablet or drageeing core.Suitable vehicle is carbohydrate or protein weighting agent, as sugar, comprises lactose, sucrose, N.F,USP MANNITOL or Sorbitol Powder; Derive from the starch of corn, wheat, paddy rice, potato or other plant; Mierocrystalline cellulose, as methylcellulose gum, Vltra tears or Xylo-Mucine; Natural gum is as gum arabic and tragacanth gum; With protein such as gelatin and collagen protein.If expectation can be added disintegrating agent or stablizer, as crosslinked polyvinylpyrrolidone, agar, Lalgine or its salt are as sodium alginate.

The drageeing core can with the dressing that is fit to, unite use as spissated sugar soln, it also can contain gum arabic, talcum, polyvinylpyrrolidone, carbopol gel, polyoxyethylene glycol and/or titanium dioxide, lacquer solution and the organic solvent or the solvent mixture that are fit to.Can in tablet or drageeing dressing, add tinting material or pigment and be used for the amount that active compound was identified or characterized to product, as dosage.

The soft seal capsule that the pharmaceutical preparation that can orally use comprises that gelatin makes pushes filling (push-fit) capsule and made by gelatin and dressing such as glycerine or sorbyl alcohol.Push filling (push-fit) and agree with capsule and can contain and weighting agent or tackiness agent such as lactose or starch, lubricant such as talcum or Magnesium Stearate and stablizer blended active ingredient alternatively.In soft capsule, can be at the suitable liquid that has or do not have stablizer, as dissolving or suspension active compound in fatty oil, liquid or the liquid macrogol.

Can be in the aqueous solution, compatible buffers on the preferred physiology is as preparing the pharmaceutical preparation that is suitable for administered parenterally in Hank ' s solution, Ringer ' s solution or the physiological buffer salt solution.Water injection suspension liquid can contain the material that increases suspension viscosity, as Xylo-Mucine, sorbyl alcohol or dextran.In addition, can be with the oily injection suspensions of suspension preparation for being fit to of active compound.The lipophilic solvent or the carrier that are fit to comprise fatty oil such as sesame oil or Acrawax such as ethyl oleate or triglyceride level or liposome.Non-lipid polycation (polycatonic) amino polymer also can be used to send.Alternatively, suspension also can comprise the reagent of suitable stablizer or increase compound dissolution to allow the preparation of highly enriched solution.

For part or nasal administration, can in preparation, utilize the permeate agent that is suitable for particular barrier to be infiltrated.This permeate agent is known in this area usually.

Can make pharmaceutical composition of the present invention with methods known in the art, for example traditional mixing, dissolving, granulation is made drageeing, grinds, and emulsification is sealed, and holds back (entrap) or freeze-drying process.

Pharmaceutical composition can be used as salt and provides, and can use many acid, includes but not limited to that hydrochloric acid, sulfuric acid, acetate, lactic acid, tartrate, oxysuccinic acid and succsinic acid wait salify.Salt tends to easier dissolving than corresponding free alkali form in water-based or other protonic solvent.In other cases, preferred preparation can be a lyophilized powder, and this lyophilized powder can contain following each or all: 1-50mM Histidine, 0.1-2% sucrose and 2-7% N.F,USP MANNITOL, pH4.5-5.5 before using mixes powder and damping fluid.

After the pharmaceutical compositions, can place them in the fitted vessel and mark on its indication that is used for the treatment of.For using of antisense nucleotide or antagonist, this mark will comprise consumption, frequency and medication.Those skilled in the art will be to antisense nucleotide and antagonist, and for example antibody or inhibitor use different preparations.For example, in U.S. Patent number 5,008,114; 5,505,962; 5,641,515; 5,681,811; 5,700,486; 5,766,633; 5,792,451; 5,853,748; 5,972,387; 5,976,569; With 6,051, the pharmaceutical preparation that is suitable for the protein oral administration has been described in 561.

On the other hand, can be by detecting the mRNA or the protein expression level of at least a open genes encoding, or the activity of proteins of at least a open genes encoding, the patient treatment that monitoring is carried out with therapeutical agent as mentioned above.These mensuration will show that whether treating is effectively or not should adjust or optimize this treatment.Therefore, during clinical trial, one or more genes described herein can be as the mark of pharmaceutical efficacy.

In useful especially embodiment, the method of monitoring medicament (as antagonist, protein, nucleic acid, small molecules or other therapeutical agent or candidate's medicament of identifying by screening method described herein) to suffering from breast cancer or having the dangerous patient's that suffers from breast cancer treatment to render a service is provided, comprised:

A) before pharmacy application, obtain sample before the administration from the patient;

B) detect before the administration in the sample by the mRNA of at least a described genes encoding or protein expression level or by the activity of proteins of at least a described genes encoding;

C) obtain sample after one or more administrations from the patient;

D) detect after the administration in the sample by the mRNA of described at least a genes encoding or protein expression level or by the activity of proteins of described at least a genes encoding;

E) with in the sample before the administration by the mRNA of described at least a genes encoding or protein expression level or by making comparisons by the mRNA of this at least a genes encoding or protein expression level or by the activity of proteins of this at least a genes encoding in the sample after the activity of proteins of described at least a genes encoding and the one or more administration; With

F) correspondingly adjust the administration of medicament.

For example, may expect to increase the medicament administration and make it more high or low, promptly increase the validity of medicament than detected level to change described at least a expression of gene level or activity.Alternately, may expect to reduce the medicament administration and make it more high or low, promptly reduce the validity of medicament than detected level to change described at least a expression of gene.

On the other hand, provide the outgrowth method of breast cancer tissue among the inhibition patient, this method has been utilized above-mentioned therapeutical agent, for example antisense nucleotide, ribozyme, double-stranded RNA and antagonist such as antibody.Utilize antisense nucleotide suppress breast cancer tissue outgrowth aspect, this method comprises the isolated nucleic acid molecule to patient's administering therapeutic significant quantity, this nucleic acid molecule comprises and derives from table 1,2, the antisense base sequences of at least a gene of identifying in 3 or 4, wherein this antisense nucleotide ability of described at least a gene transcription/translation that changes.

Utilize ribozyme suppress breast cancer tissue outgrowth aspect, this method comprises the nucleotide sequence to the encoding ribozyme of patient's administering therapeutic significant quantity, the nucleotide sequence of this encoding ribozyme table 1 that changes, the ability of at least a gene transcription/translation of identifying in 2,3 or 4.

Utilize double-stranded RNA suppress breast cancer tissue outgrowth aspect, this method comprise to patient's administering therapeutic significant quantity corresponding to table 1, the double-stranded RNA of at least a gene of identifying in 2,3 or 4, wherein the double-stranded RNA ability of described at least a gene transcription/translation that changes.

Utilize antagonist suppress breast cancer tissue outgrowth aspect, this method comprises the antagonist to patient's administering therapeutic significant quantity, this antagonist can cause table 1, the proteinic inhibition or the activation of at least a coded by said gene of identifying in 2,3 or 4.

Suppressing under the outgrowth situation of breast cancer tissue, " the treatment significant quantity " of nucleotide sequence, double-stranded RNA or antagonist that comprises isolated nucleic acid molecule, the encoding ribozyme of antisense nucleotide refers to that one of these healing potions (for example are enough to suppress breast cancer tissue's hyperplasia, the cell growth of inhibition or stable breast cancer tissue) amount, and can measure as mentioned above.

The purposes of virus vector

In yet another aspect, the virus vector that comprises following gene promoter is provided, this gene is selected from table 1,2, at least a gene of identifying in 3 or 4, the wherein said promotor carrier that is operably connected duplicates necessary gene coding region, and wherein this carrier is adapted to duplicate after transfection enters mammary gland cell.

This carrier can be in mammary tissue and copy choice in non-mammary tissue not.Duplicating there to be positive transcription factor in the mammary tissue but not having this positive transcription factor in the mammary tissue is condition, and this positive transcription factor will activate the promotor of open gene.Normally appear at transcription inhibition factor that stops promoter transcription in the non-mammary tissue owing to lack, also can occur duplicating.Therefore, when transcribing when taking place, it continues to enter and duplicates essential gene, therefore, and in mammary tissue and in non-mammary tissue, do not go out the function of following with it of duplicating of expression vector.Utilize this carrier can the ill mammary tissue of selective therapy, breast tumor for example, and have minimum system toxicity.

In one embodiment, virus vector is an adenovirus carrier, and it comprises carrier and duplicates necessary gene coding region, and wherein the coding region is selected from: E1a, E1b, E2 and E4 coding region.The method for preparing this carrier is known to those of ordinary skills, referring to for example, and Sambrook etc., " Molecular Cloning:A Laboratory Manual ", and Cold Spring Harbor, NY (1989) is described.

In an embodiment again, the vector encoded heterologous gene products, this heterologous gene products will be from this vector expression in mammary gland cell.Heterologous gene products provides ill mammary tissue, for example inhibition, prevention or the destruction of breast tumor growth.

Gene product can be RNA, for example sense-rna or ribozyme, or protein, and as cytokine, interleukin-for example, Interferon, rabbit or toxin such as diphtheria toxin, pseudomonal toxin etc.Heterologous gene products also can be negative selective key thing such as Isocytosine deaminase.This negative selective key thing can interact to stop, to suppress or destroy the growth of ill mammary gland cell with other medicament.

Carrier transfection of the present invention can be entered the auxiliary cell line that is used for virus replication and produces infectious viral particle.Alternately, can pass through electroporation, calcium phosphate precipitation, microinjection or the carrier transfection is entered mammary gland cell by proteoliposome (Proteoliposome).At for example U.S. Patent number 5,998, describe the method and their purposes in tumour cell and the treatment of other type abnormal cells that prepare the tissue specificity replicating vector in 205 in detail, described tumour cell and other type abnormal cells are deleterious or undesirable in patient's body.

The nucleic acid of thing and proteinic detection as a token of

In a particularly embodiment, utilize methods known in the art, by in biological sample, detecting level corresponding to the mRNA of mark with original position and external mode.Term " biological sample " means and comprises tissue, cell and the liquid that exists in the isolating tissue of patient, cell, biological liquid and its isolate and the patient's body.Many detection of expression methods are utilized isolating RNA.For in vitro method, any can optionally not resist the purifying that the isolating RNA isolation technique of mRNA may be used to mammary gland cell RNA (referring to, editor such as Ausubel for example, " Current Protocols in MolecularBiology ", John Wiley ﹠amp; Sons, NY (1987-1999)).In addition, utilize the technology that well known to a person skilled in the art, as, Chomczynski, U.S. Patent number 4,843,155 (1989) single step RNA separation method can easily be handled a large amount of tissue samples.

Isolating mRNA can be used in hybridization or the amplification assay, and described mensuration includes but not limited to that Southern or Northern analyze, and the polymerase chain reaction is analyzed and probe assay.A kind of diagnostic method that preferably is used for the mRNA level detection comprises makes isolating mRNA contact with nucleic acid molecule (probe), and this nucleic acid molecule can be hybridized with the mRNA of genes encoding to be detected.Nucleic acid probe can be, for example full-length cDNA or its part, as grow to few 7,15,30,50,100,250 or 500 Nucleotide and under stringent condition, being enough to specifically and the mRNA of code book invention mark or the oligonucleotide of genomic dna hybridization.Here also describe other and be useful in suitable probe in the diagnostic assay of the present invention.The hybridization of mRNA and probe shows expresses described mark.

In one form, by for example isolating mRNA of electrophoresis on sepharose, shift mRNA to film from gel then, on nitrocellulose filter, fixedly mRNA contacts on solid phase carrier and with probe.In alternative form, for example in Affymetrix gene chip array, stationary probe and makes mRNA contact with probe on solid phase carrier.Those skilled in the art can easily revise known mRNA detection method with in the level that is used in the mRNA that detects mark coding of the present invention.

Be used for test sample and relate to amplification process corresponding to the alternative of the mRNA level of mark of the present invention, for example, by rtPCR (seeing Mullis, U.S. Patent number 4,683, the experiment embodiment described in 202 (1987)); Ligase chain reaction (LCR) (Barany, Proc.Natl.Acad.Sci.USA, 88 volumes, 189-193 page or leaf (1991)); Automatically keep sequence replicating (Guatelli etc., Proc.Natl.Acad.Sci.USA, 87 volumes, 1874-1878 page or leaf (1990)); Transcription amplification system (Kwoh etc., Proc.Natl.Acad.Sci.USA, 86 volumes, 1173-1177 page or leaf (1989)); Q-β replicative enzyme (Lizardi etc., Bio/Technology, 6 volumes, 1197 pages (1988)); Rolling-circle replication (Lizardi etc., U.S. Patent number 5,854,033 (1988)); Or any other nucleic acid amplification method amplification process of carrying out, then utilize the molecule that well known to a person skilled in the art the technology for detection amplification.If nucleic acid molecule occurs with low-down quantity, these detection schemes are particularly useful for the detection of this nucleic acid molecule.As used herein, amplimer be defined as can with a pair of nucleic acid molecule of gene (be respectively the positive and negative chain, or vice versa) 5 ' or 3 ' regional annealing, contain short zone between the two.Usually, amplimer is 10-30 length of nucleotides approximately, and flank is the zone of about 50-200 length of nucleotides.In the condition that is fit to have under the situation of suitable reagent, this primer allows to comprise the amplified nucleic acid molecule of the nucleotide sequence that primer surrounds.

For in-situ method, do not need from the mammary gland cell separating mRNA before the detection.In this method, with known Histological method preparation/processing cell or tissue sample.At carrier, normally fixed sample on the slide glass contacts with probe then then, and this probe can be hybridized with the mRNA of coding maker thing.

Replacement scheme as the absolute expression levels based on mark detects can detect based on the mark expression level of mark.Expression by mark relatively and be not the gene of mark is for example formed the absolute expression levels of expression correction mark of the housekeeping gene of expression and is come the normalized expression level.Be used for standardized suitable gene and comprise the gene that housekeeping gene such as actin gene or epithelial cell are special.This stdn allows a sample, for example expression level in patient's sample and another sample, and the expression level in for example non-mammary cancer sample compares, or permission compares at the sample room of different sources.

Alternately, can provide expression level with the form of relative expression's level.In order to determine relative expression's level of mark, before the expression level that detects described sample, detects 10 or more, preferred 50 or more a plurality of normally and the marker expression level of the isolate sample of cancer cells.Determine the average expression level of each gene of in a large amount of samples, measuring, and with this baseline expression level of thing as a token of.The marker expression level (absolute expression levels) of using the test sample of measuring then is divided by the average expression values that obtains at this mark.This provides the relative expression level.

Preferably, be used in the non-breast cancer cell that sample in the baseline determination will derive from mammary cancer or derive from mammary tissue.The application of relative expression's level is depended in the selection in cell source.Utilize the expression in the healthy tissues can help to verify that as the average mark of expressing the mark of whether measuring is mammary gland special (with respect to a normal cell).In addition,, can revise average expression values, provide improved relative expression's value according to the data that accumulate along with more multidata accumulation.The means of classification breast cancer status seriousness are provided from the expression data of mammary gland cell.

In another embodiment of the invention, detect polypeptide corresponding to mark.The preferred reagent that detects polypeptide of the present invention is preferably to have the antibody of detectable label in conjunction with the antibody of the polypeptide of mark correspondence of the present invention.Antibody can be polyclone, or monoclonal antibody more preferably.Can utilize complete antibody, or its fragment (for example Fab or F (ab ') ₂).The term " mark " that relates to probe or antibody is intended to comprise by coupling (promptly, physical bond) detectable material on probe or the antibody to probe or the direct mark of antibody, and by with the another kind of reagent react of direct mark to probe or antibody indirect mark.The example of indirect labelling comprises and utilizes fluorescently-labeled second antibody to detect first antibody and with the end-labelled dna probe of fluorescently-labeled streptavidin detection of biological element.

Utilization well known to a person skilled in the art that technology can be from the mammary gland cell isolated protein.Can use, for example, at Harlow and Lane, " Antibodies:A Laboratory Manual ", Harlow and Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, the method for protein isolation described in the NY (1988).

Can utilize various forms to detect sample whether and contain protein in conjunction with given antibody.The example of these forms includes but not limited to: enzyme immunoassay (EIA); Radioimmunoassay (RIA), Western engram analysis and ELISA.Those skilled in the art can easily revise known protein matter/antibody detection method and detect to be used in whether mammary gland cell is expressed in the method for mark of the present invention.

In one form, antibody or antibody fragment can be used in method, as in Western trace or the immunofluorescence technique to detect expressed protein.In this application, general preferred on solid support sessile antibody or protein.Solid support that is fit to or carrier comprise any support of energy conjugated antigen or antibody.Known support or carrier comprise glass, Mierocrystalline cellulose, polyacrylamide, gabbro and the magnetite of polystyrene, polypropylene, polyethylene, dextran, nylon, amylose starch, natural and modification.

Those skilled in the art will know that many other be used for binding antibody or antigenic suitable carrier, and can this support of adaptability revision to use with the present invention.For example, from the isolating protein of mammary gland cell can be on polyacrylamide gel electrophoresis, and be fixed on solid support such as the nitrocellulose.Wash support with the damping fluid that is fit to then, then use the antibody treatment of detectable label.Then can be with the support of damping fluid secondary washing solid phase to remove unconjugated antibody.Can detect bonded labelled amount on the solid support by ordinary method then.

The present invention also comprises and being used in the detection of biological sample (for example, the relevant body fluid of mammary gland, serum, blood plasma, lymph, gall-bladder liquid, urine, ight soil, CSF, ascites or blood) corresponding to the polypeptide of mark of the present invention or the test kit of nucleic acid existence.This test kit can be used to detect whether the patient suffers from mammary cancer, perhaps is in the increase danger of suffering from mammary cancer.For example, this test kit can comprise can the detection of biological sample in corresponding to tagged compound or the reagent of the mRNA of the polypeptide of mark of the present invention or coded polypeptide, means (for example, in conjunction with the antibody of polypeptide or in conjunction with the DNA of coded polypeptide or the oligonucleotide probe of mRNA) with polypeptide in the working sample or mRNA amount.Test kit also can comprise the specification sheets of explaining with this test kit gained result.

For test kit based on antibody, test kit can comprise, for example 1) can be in conjunction with (for example corresponding to the first antibody of the polypeptide of mark of the present invention, be connected with solid support), alternatively 2) the second different antibody, this second antibody can be puted together in conjunction with polypeptide or first antibody and with detectable label.

For the test kit based on oligonucleotide, this test kit can comprise, for example 1) can with the oligonucleotide of coding corresponding to the nucleic acid array hybridizing of the polypeptide of mark of the present invention, for example oligonucleotide of detectable label; Perhaps 2) a pair ofly can be used for increasing corresponding to the primer of the nucleic acid molecule of mark of the present invention.This test kit also can comprise, for example buffer reagent, sanitas or protein stabilizing agent.Test kit can further comprise the detection necessary component of detectable label (for example enzyme or substrate).Test kit also can comprise control sample or serial control sample, can measure these control samples and with test sample relatively.

Every kind of component of test kit can be contained in the independent container, and these all various containers can wrap in the individual packaging with the specification sheets of the measurement result of explaining this test kit.

The monitoring clinical trial

Monitoring reagent (for example pharmaceutical composition) not only can be applied in the essential drugs screening the influence of marker expression level of the present invention, also can be applied in the clinical trial.For example, can be in the patient's who accepts breast cancer treatment clinical trial monitoring reagent influence the validity of marker expression.In preferred embodiments, the invention provides the method for monitoring with medicament (for example agonist, antagonist and polypeptide simulation, protein, peptide, nucleic acid, small molecules or other medicines material standed for) treatment patient's validity, comprise following step:

(i) before using medicament, obtain sample before the administration from the patient;

(ii) detect the expression level of one or more selected marks of the present invention in the preceding sample of administration;

(iii) obtain sample after one or more administrations from the patient;

(iv) detect after the administration expression level of this mark in the sample;

(v) the marker expression level in the sample after the marker expression level in the sample before the administration and the one or more administration is compared; With

(vi) correspondingly change patient's pharmacy application scheme;

For example, may expect to increase the medicament administration and make it be higher than detected level, promptly increase the validity of medicament with the expression that increases mark.Alternately, may expect to reduce the administration of medicine reagent to reduce the validity of medicament.

Experimental program

Subtracted library and making transcript spectrum

Utilize the method for PCR-based to produce subtracted library, this method allows to isolate in a mRNA group (tester) than the clone who expresses with higher level in another group's (driver).By reverse transcription tester and driver mRNA group are converted to cDNA, use the SMART of Clontech then ^TMThe PCR test kit carries out pcr amplification.PCR-with Clontech selects cDNA deduction test kit to make the cDNA hybridization of tester and driver then.This technology causes deduction and stdn,, makes the copy number equalization of low abundance and high abundance sequence that is.After subtracted library produces, detect one group 96 of each library or more a plurality of clone with the confirmation differential expression with anti-phase Southern hybridization.

For the mark of identifying by above-mentioned subtracted library hybridization technique of the present invention, " tester " of subtracted library source is by forming from 3 kinds of mammary cancer (obtaining from people patient) tissue sample or the cDNA that produces from breast cancer cell line." driver " source of subtracted library is made up of the cDNA that produces from non-carcinous mammary tissue cell.

For the transcript spectrum, utilize the robot grid system that is connected with the sample data storehouse, by preparing the nylon array at the PCR product of putting purifying on the nylon membrane.Can put several thousand clones on each nylon leaching film.

Be used to RNA or DNA hybridization nylon array from (tumour and normal) clinical sample and clone.This RNA or DNA utilize external reverse transcription reaction mark, and this external reverse transcription reaction comprises the radiolabeled oligonucleotide that can during reaction mix.Alternately, mRNA is converted into cDNA, utilizes the SMART PCR test kit of Clontech to carry out pcr amplification then by reverse transcription.Carry out cross experiment by in hybridization chamber, the RNA of mark or DNA sample being mixed with nylon leaching film.Carry out two parts of multiple independence cross experiments and transcribe spectrum data (referring to, NatureGenetics, 21 volumes (1999)) with generation.

The reference of quoting

Incorporating all documents that this paper quotes into for all purpose integral body here is reference, and this incorporating into just is equal in detail that to incorporate every piece of publication or patent or patent application into be that reference is the same with being indicated as being all purpose integral body individually, in addition, be incorporated herein all GenBank accession number of quoting, single-gene bunch number and protein accession number here for all purposes are whole as a reference, and this incorporating into is equal to that to incorporate each this numbering into the same as a reference with indicating integral body individually in detail for all purposes.

The invention is not restricted to the particular that the application describes, these particular are intended to illustrate as the single of each side of the present invention.Can carry out many modifications and change to the present invention, and not deviate from the spirit and scope of the present invention, this will be conspicuous to those skilled in the art.Except the method and instrument enumerated here, from aforementioned description and accompanying drawing, those skilled in the art will understand that functional equivalent processes and instrument are also within the scope of the present invention.This modification and change are intended to fall in the scope of claims.The present invention only is subject to the four corner of the appended equivalent that claim and this claim contained.

Claims (81)

1, a kind of ill object of mammary cancer that screens comprises to predict the method for replying of described mammary cancer to endocrine therapy:

A) detect available from the breast tumor biopsy of object corresponding to the mRNA expression level of gene NOVA1, to obtain first value;

B) detect available from its tumour and endocrine therapy is produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain second value corresponding to gene NOVA1;

C) detect available from its tumour and endocrine therapy is not produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain the 3rd value corresponding to gene NOVA1; With

D) relatively first value and the second and the 3rd value, wherein first value is similar to second value and indicate than the 3rd value height that the tumour of object will produce endocrine therapy and reply; And wherein first value is littler and similarly to the 3rd value show that object will not produce endocrine therapy and reply than second value.

2, a kind of ill object of mammary cancer that screens comprises to predict the method for replying of described mammary cancer to endocrine therapy:

A) detect available from the breast tumor biopsy of object corresponding to the mRNA expression level of gene IGHG3, to obtain first value;

B) detect available from its tumour and endocrine therapy is produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain second value corresponding to gene IGHG3;

C) detect available from its tumour and endocrine therapy is not produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain the 3rd value corresponding to gene IGHG3;

D) relatively first value and the second and the 3rd value, wherein first value is similar to second value and indicate than the 3rd value height that the tumour of object will produce endocrine therapy and reply; And wherein first value is littler and similarly to the 3rd value show that object will not produce endocrine therapy and reply than second value.

3, a kind of ill object of mammary cancer that screens comprises to predict the method for replying of described mammary cancer to endocrine therapy:

A) detect available from the breast tumor biopsy of object corresponding to the mRNA expression level of genes identified at least a table 3, to obtain first value;

B) detect available from its tumour and endocrine therapy is produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain second value corresponding at least a genes identified described in a);

C) detect available from its tumour and endocrine therapy is not produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain the 3rd value corresponding at least a genes identified described in a);

D) relatively first value and the second and the 3rd value, wherein first value is similar to second value and indicate than the 3rd value height that the tumour of object will produce endocrine therapy and reply; And wherein first value is littler and similarly to the 3rd value show that object will not produce endocrine therapy and reply than second value.

4, a kind of ill object of mammary cancer that screens comprises to predict the method for replying of described mammary cancer to endocrine therapy:

A) detect available from the breast tumor biopsy of object corresponding to the mRNA expression level of genes identified at least a table 4, to obtain first value;

B) detect available from its tumour and endocrine therapy is produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain second value corresponding at least a genes identified described in a);

C) detect available from its tumour and endocrine therapy is not produced in the patient's who replys the breast tumor biopsy mRNA expression level, to obtain the 3rd value corresponding at least a genes identified described in a);

D) relatively first value and the second and the 3rd value, wherein first value is similar to second value and hang down the tumour that indicates object than the 3rd value and will produce endocrine therapy and reply; And wherein first value is similar to the 3rd value and will not produce endocrine therapy than the high tumour that indicates object of second value and reply.

5, a kind of method for the treatment of mammary cancer, comprise: give the synthesizing of gene product that the patient of this treatment of needs uses one or more genes shown in can reconciliation statement 1,2,3 or 4 or these genes, expression or active compound, to improve at least a symptoms of breast cancer.

6, method as claimed in claim 5, wherein said gene is selected from: non-voltage-gated sodium channel 1 α (SCNN1A); Serine or cystatin clade A member 3 (SERPINA3); N-acyl sphingosine hydroamidase (ASAH); Lipocalin 1 (LCN1); The III receptor (TGFBR3) of transforming growth factor-beta; IIB subtribe (CYP2B), AZGP1, NOVA1 and the IGHG3 of glutamate receptor precursor 2 (GRIA2) and phenylethyl barbituric acid inductive Cytochrome P450.

7, method as claimed in claim 5, wherein said gene product is selected from the protein of following genetic expression: non-voltage-gated sodium channel 1 α (SCNN1A); Serine or cystatin; Clade A member 3 (SERPINA3); N-acyl sphingosine hydroamidase (ASAH); Lipocalin 1 (LCN1); The III receptor (TGFBR3) of transforming growth factor-beta; IIB subtribe (CYP2B), AZGP1, NOVA1 and the IGHG3 of glutamate receptor precursor 2 (GRIA2) and phenylethyl barbituric acid inductive Cytochrome P450.

8, the method for a kind of definite breast tumor to whether generation being replied based on endocrine treatment comprises:

A) in the check breast tumor tissues sample corresponding to the mRNA expression level of genes identified at least a table 1,2,3 or 4, so that first value to be provided;

B) detect available from the mammary tissue sample of non-diseased individuals corresponding to the mRNA expression level of genes identified at least a table 1,2,3 or 4, so that second value to be provided; With

C) relatively first value and second value, wherein first value than the high individuality of suffering from breast tumor that shows of second value to generation being replied based on endocrine treatment.

9, whether a kind of mammary cancer of definite object will comprise producing the method for replying based on endocrine treatment:

A) detection is available from the expression level of the gene expression product of NOVA1 gene in the ill sample of object, to obtain first value;

B) detection is available from the expression level of the gene expression product of NOVA1 gene in its tumour patient's that generation is replied to endocrine therapy the ill sample, to obtain second value;

C) detect the expression level that endocrine therapy is not produced the gene expression product of NOVA1 gene in the patient's who replys the ill sample available from its tumour, to obtain the 3rd value; With

D) relatively first value and the second and the 3rd value, wherein first value and ratio three value height similar to second value shows that the tumour of object will produce endocrine therapy and replys; And wherein first value low and similar tumour that shows object to the 3rd value will not produce endocrine therapy and reply than second value.

10, method as claimed in claim 9 wherein replaces the NOVA1 gene, detects the expression level of the gene product of IGHG3 gene.

11, as claim 9 or 10 described methods, wherein ill sample is the relevant body sample of mammary gland, is selected from: breast biopsy tissue, blood, serum, blood plasma, lymph, ascites, gall-bladder liquid, urine, CSF, mammary gland transudate or nipple aspirate.

12, as the described method of claim 9,10 or 11, wherein the expression level of gene expression product is assessed corresponding to the proteinic existence of this gene expression product by detecting.

13, method as claimed in claim 12 wherein utilizes the proteinic reagent of specific combination to detect proteinic existence.

14, method as claimed in claim 13, wherein reagent is selected from: antibody, antibody derivatives and antibody fragment.

15, a kind of mammary cancer that is used to detect patient whether will be to producing the test kit of replying based on endocrine treatment, and it is included in claim 13 in the container that is suitable for contacting the relevant body fluid of mammary gland or 14 reagent.

16, test kit as claimed in claim 15, wherein said reagent comprises antibody, wherein said antibody is specifically in conjunction with the protein corresponding to gene expression product of claim 12.

17, a kind of method for the treatment of patient's mammary cancer, comprise synthetic, expression or the active compound of using one or more genes that to regulate in a group gene or gene expression product to described patient, to improve at least a symptoms of breast cancer, wherein said gene group comprises those genes of identifying in the table 1,2,3 or 4.

18, method as claimed in claim 17, wherein compound is selected from: antisense molecule, double-stranded RNA, ribozyme, micromolecular compound, antibody or antibody fragment.

19, monitoring suffer from or the dangerous patient who suffers from mammary cancer in the method for mammary cancer progress, comprise: As time goes on, measurement available from patient's body fluid or the mammary tissue sample corresponding to the mRNA expression level of at least a gene in a group gene, wherein said gene group comprises those genes of identifying in the table 1,2,3 or 4, wherein As time goes on, the increase of the mRNA expression level of described at least a gene shows the progress of mammary cancer among the patient.

20, method as claimed in claim 19, genes identified is selected from the wherein said at least a table 1,2,3 or 4: TFF1, TFF3, SERPINA3, PIP, MGP, TGFRB3 and AZGP1.

21, method as claimed in claim 19, the wherein expression level of the technology for detection mRNA by being selected from Northern engram analysis, reverse transcription PCR and real-time quantitative PCR.

22, monitoring suffer from or the dangerous patient who suffers from mammary cancer in the method for mammary cancer progress, comprise: As time goes on, measurement available from patient's body fluid or the mammary tissue sample by the expression level of genes identified encoded protein matter at least a table 1,2,3 or 4, wherein As time goes on, the increase of the protein expression level of described at least a genes encoding shows the progress of mammary cancer among the patient.

23, method as claimed in claim 22, genes identified is selected from the wherein said at least a table 1,2,3 or 4: TFF1, TFF3, SERPINA3, PIP, MGP, TGFRB3 and AZGP1.

24, monitoring suffer from or the dangerous patient who suffers from mammary cancer in the method for mammary cancer progress, comprise: As time goes on, measurement available from patient's body fluid or the mammary tissue sample corresponding to the mRNA expression level of at least a gene that is selected from those genes of identifying in the table 1,2,3 or 4, wherein As time goes on, the change of the mRNA expression level of described at least a gene shows the progress of mammary cancer among the patient.

25, monitoring suffer from or the dangerous patient who suffers from mammary cancer in the method for mammary cancer progress, comprise: As time goes on, measurement is available from the protein expression level that is selected from least a coded by said gene of those genes of identifying in the table 1,2,3 or 4 in patient's body fluid or the mammary tissue sample, wherein As time goes on, the change of the protein expression level of described at least a genes encoding shows the progress of mammary cancer among the patient.

26, method as claimed in claim 25, the protein expression level of wherein said at least a genes encoding are utilized the special label probe of this protein are detected by the Western trace.

27, method as claimed in claim 26, wherein label probe is an antibody.

28, method as claimed in claim 27, wherein antibody is monoclonal antibody.

29, a kind of evaluation can be used for the method for the medicament of breast cancer treatment, and it is made up of following steps:

A) with the mammary tissue sample of candidate's medicament contact available from the mammary cancer suspected patient;

B) the mRNA expression level of at least a gene in the test sample, wherein said at least a gene are selected from the gene group that comprises those genes of identifying in the table 1,2,3 or 4; With

C) will compare at mRNA expression level of at least a gene described in the sample under the situation that candidate's medicament exists and the mRNA expression level of at least a gene described in the sample under the situation of candidate's medicament shortage, wherein with respect to the mRNA expression level of this at least a gene in the sample under the situation about lacking at candidate's medicament, the reduction or the increase of the mRNA expression level of this at least a gene show that medicament is useful in the sample in breast cancer treatment under the situation that candidate's medicament exists.

30, method as claimed in claim 29, genes identified is selected from the wherein said at least a table 1,2,3 or 4: TFF1, TFF3, SERPINA3, PIP, MGP, TGFRB3 and AZGP1.

31, method as claimed in claim 29, the wherein expression level of the technology for detection mRNA by being selected from Northern engram analysis, reverse transcription PCR and real-time quantitative PCR.

32, method as claimed in claim 29, wherein medicament is selected from: small molecules and antisense polynucleotides.

33, a kind of evaluation can be used for the method for the medicament of breast cancer treatment, and it is made up of following steps:

A) with body fluid or the mammary tissue sample of candidate's medicament contact available from the mammary cancer suspected patient;

B) the protein expression level of at least a genes encoding in the test sample, wherein said at least a gene are selected from the gene group that comprises those genes of identifying in the table 1,2,3 or 4;

C) will compare in protein expression level of at least a genes encoding described in the sample under the situation that candidate's medicament exists and the protein expression level of at least a genes encoding described in the sample under the situation of candidate's medicament shortage, wherein with respect to the protein expression level of this at least a genes encoding in the sample under the situation about lacking at candidate's medicament, the reduction or the increase of the protein expression level of this at least a genes encoding show that medicament is useful in the sample in breast cancer treatment under the situation that candidate's medicament exists.

34, method as claimed in claim 33, the wherein said at least a gene that is selected from the gene group that comprises those genes of identifying in the table 1,2,3 or 4 is: TFF1, TFF3, SERPINA3, PIP, MGP, TGFRB3 and AZGP1.

35, method as claimed in claim 33, the protein expression level of wherein said at least a genes encoding are utilized the special label probe of this protein are detected by the Western trace.

36, method as claimed in claim 35, wherein label probe is an antibody.

37, method as claimed in claim 36, wherein antibody is monoclonal antibody.

38, a kind of evaluation can be used for the method for the medicament of breast cancer treatment, comprising:

A) with the mammary tissue sample of candidate's medicament contact available from the mammary cancer suspected patient;

B) the mRNA expression level of at least a gene in the test sample, wherein said gene are selected from those genes of identifying in the table 1,2,3 or 4; With

C) will compare at mRNA expression level of at least a gene described in the sample under the situation that candidate's medicament exists and the mRNA expression level of at least a gene described in the sample under the situation of candidate's medicament shortage, wherein with respect to the mRNA expression level of this at least a gene in the sample under the situation about lacking at candidate's medicament, the change of the mRNA expression level of this at least a gene shows that medicament is useful in the sample in breast cancer treatment under the situation that candidate's medicament exists.

39, method as claimed in claim 38, the wherein expression level of the technology for detection mRNA by being selected from Northern engram analysis, reverse transcription PCR and real-time quantitative PCR.

40, method as claimed in claim 41, wherein medicament is selected from small molecules and antisense polynucleotides.

41, a kind of evaluation can be used for the method for the medicament of breast cancer treatment, comprising:

A) with body fluid or the mammary tissue sample of candidate's medicament contact available from the galactophore disease suspected patient;

B) the protein expression level of at least a genes encoding in the test sample, wherein said gene are selected from those genes of identifying in the table 1,2,3 or 4; With

C) will compare in protein expression level of at least a genes encoding described in the sample under the situation that candidate's medicament exists and the protein expression level of at least a genes encoding described in the sample under the situation of candidate's medicament shortage, wherein with respect to the protein expression level of this at least a genes encoding in the sample under the situation about lacking at candidate's medicament, the change of the protein expression level of this at least a gene shows that medicament is useful in the sample in breast cancer treatment under the situation that candidate's medicament exists.

42, method as claimed in claim 41, the protein expression level of wherein said at least a genes encoding are utilized the special label probe of this protein are detected by the Western trace.

43, method as claimed in claim 41, wherein label probe is an antibody.

44, method as claimed in claim 43, wherein antibody is monoclonal antibody.

45, method as claimed in claim 41, wherein medicament is selected from small molecules and antisense polynucleotides.

46, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, comprise isolated nucleic acid molecule to patient's administering therapeutic significant quantity, this isolated nucleic acid molecule origin comes from the antisense base sequences of at least a gene that is selected from those genes of identifying in the table 1,2,3 or 4 to be formed, and can change described at least a gene transcription/translation.

47, method as claimed in claim 46, wherein said at least a gene is selected from: TFF1, TFF3, SERPINA3, PIP, MGP, TGFRB3 and AZGP1.

48, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, comprise the antagonist to patient's administering therapeutic significant quantity, this antagonist can suppress/activate the protein by at least a genes encoding that is selected from those genes of identifying in the table 1,2,3 or 4.

49, method as claimed in claim 48, wherein said at least a gene is selected from: TFF1, TFF3, SERPINA3, PIP, MGP, TGFRB3 and AZGP1.

50, method as claimed in claim 48, wherein antagonist is the antibody special to described protein.

51, method as claimed in claim 50, wherein antibody is monoclonal antibody.

52, method as claimed in claim 51, wherein monoclonal antibody and toxic agents are puted together.

53, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, it is made up of following steps: the isolated nucleic acid molecule of giving patient's administering therapeutic significant quantity, this isolated nucleic acid molecule origin comes from the antisense base sequences of at least a gene that is selected from those genes of identifying in the table 1,2,3 or 4 to be formed, and can reduce/increase described at least a gene transcription/translation.

54, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, it comprises the antagonist to patient's administering therapeutic significant quantity, and this antagonist can suppress/activate the protein by at least a genes encoding that is selected from those genes of identifying in the table 1,2,3 or 4.

55, method as claimed in claim 54, wherein antagonist is the antibody special to described protein.

56, method as claimed in claim 55, wherein antibody is monoclonal antibody.

57, method as claimed in claim 56, wherein monoclonal antibody and toxic agents are puted together.

58, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, comprise the nucleotide sequence to the encoding ribozyme of patient's administering therapeutic significant quantity, this nucleotide sequence can reduce/increase at least a gene transcription/translation that is selected from those genes of identifying in the table 1,2,3 or 4.

59, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, comprise the double-stranded RNA corresponding at least a gene of definition in the claim 58 to patient's administering therapeutic significant quantity, this double-stranded RNA can reduce this at least a gene transcription/translation.

60, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, comprise the nucleotide sequence to the encoding ribozyme of patient's administering therapeutic significant quantity, this nucleotide sequence can change at least a gene transcription/translation that is selected from those genes of identifying in the table 1,2,3 or 4.

61, a kind of treatment suffers from or the dangerous method of suffering from the patient of mammary cancer, comprise double-stranded RNA, the change ability of this at least a gene transcription/translation of this double-stranded RNA corresponding at least a gene that is selected from the table 1,2,3 or 4 those genes of identifying to patient's administering therapeutic significant quantity.

62, a kind ofly be used to monitor medicament to the method for suffering from or the dangerous treatment of suffering from the patient of mammary cancer is renderd a service, this method comprises:

A) give with medicament before obtain administration from the patient before sample;

B) detect corresponding to the mRNA expression level that is selected from the gene of those genes of evaluation in the table 1,2,3 or 4;

C) obtain sample after one or more administrations from the patient;

D) detect after these one or more administrations in the sample mRNA expression level corresponding to described at least a gene;

E) will compare corresponding to the mRNA expression level corresponding to this at least a gene in the sample after the mRNA expression level of this at least a gene and the administration in the sample before the administration;

F) correspondingly adjust the dosage regimen of medicament.

63, a kind ofly be used to monitor medicament to the method for suffering from or the dangerous treatment of suffering from the patient of mammary cancer is renderd a service, this method comprises:

A) give with medicament before obtain administration from the patient before sample;

B) detect the protein expression level that is selected from least a genes encoding of those genes of evaluation in the table 1,2,3 or 4;

C) obtain sample after one or more administrations from the patient;

D) detect the protein expression level of at least a genes encoding described in the sample after these one or more administrations;

E) the protein expression level with this at least a genes encoding in the sample after the protein expression level of this at least a genes encoding in the sample before the administration and the administration compares;

F) correspondingly adjust the dosage regimen of medicament.

64, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the isolated nucleic acid molecule to patient's administering therapeutic significant quantity, this isolated nucleic acid molecule origin comes from the antisense base sequences of at least a gene that is selected from those genes of identifying in the table 1,2,3 or 4 to be formed, and can change described at least a gene transcription/translation.

65, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the isolated nucleic acid molecule to patient's administering therapeutic significant quantity, this isolated nucleic acid molecule origin comes from the antisense base sequences of at least a gene that is selected from those genes of identifying in the table 1,2,3 or 4 to be formed, and can change described at least a gene transcription/translation.

66, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the nucleotide sequence to the encoding ribozyme of patient's administering therapeutic significant quantity, and this nucleotide sequence can change at least a gene transcription/translation that is selected from those genes of identifying in the table 1,2,3 or 4.

67, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the nucleotide sequence to the encoding ribozyme of patient's administering therapeutic significant quantity, and this nucleotide sequence can change at least a gene transcription/translation that is selected from those genes of identifying in the table 1,2,3 or 4.

68, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the double-stranded RNA corresponding at least a gene that is selected from the table 1,2,3 or 4 those genes of identifying to patient's administering therapeutic significant quantity, the change ability of this at least a gene transcription/translation of this double-stranded RNA.

69, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the double-stranded RNA corresponding at least a gene that is selected from the table 1,2,3 or 4 those genes of identifying to patient's administering therapeutic significant quantity, the change ability of this at least a gene transcription/translation of this double-stranded RNA.

70, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the antagonist to patient's administering therapeutic significant quantity, this antagonist can suppress/activate the protein by at least a genes encoding that is selected from those genes of identifying in the table 1,2,3 or 4.

71, as the described method of claim 70, wherein antagonist is the antibody special to described protein.

72, as the described method of claim 71, wherein antibody is monoclonal antibody.

73, as the described method of claim 72, wherein monoclonal antibody and toxic agents are puted together.

74, the outgrowth method of a kind of inhibition patient breast cancer tissue, this method comprises the antagonist to patient's administering therapeutic significant quantity, this antagonist can suppress the protein by at least a genes encoding that is selected from those genes of identifying in the table 1,2,3 or 4.

75, as the described method of claim 74, wherein antagonist is the antibody special to described protein.

76, as the described method of claim 75, wherein antibody is monoclonal antibody.

77, as the described method of claim 76, wherein monoclonal antibody and toxic agents are puted together.

78, a kind of virus vector, it comprises: the promotor that is selected from least a gene of those genes of identifying in the table 1,2,3 or 4, this promotor is operationally duplicated necessary gene coding region with carrier and is connected, and wherein said carrier is adapted to duplicate after transfection enters in the mammary gland cell.

79, as the described carrier of claim 78, wherein virus vector is an adenovirus carrier.

80, as carrier as described in the claim 78, wherein carrier duplicates necessary gene coding region and is selected from E1a, E1b, E2 and E4 coding region.

81, as the described carrier of claim 78,79 or 80, it also comprises the nucleotide sequence of code separated source gene product.